Methods, compositions and systems for protein detection

Abstract

The present disclosure relates to proteomics, including the detection of immune cell target proteins as examples that the disclosed methods can be used for protein detection. Methods, compositions, and systems are described for identifying the presence of proteins, including detecting immune cell proteins from a sample using a combination of multiplex immuno-PCR with antibodies conjugated to oligonucleotides comprising an antibody-specific barcode flanked by PCR priming sites; detection of barcoded amplicons obtained from immuno-PCR with encoded padlock probes comprising codes specific for the barcodes / target protein; rolling circle amplification (RCA) of the circularized padlock probes; and determination of the sequence of the codes amplified by RCA by next generation sequencing or using fluorescently labeled hybridization probes.

Description

[0001] METHODS, COMPOSITIONS AND SYSTEMS FOR PROTEIN DETECTION

[0002] CROSS-REFERENCE

[0003]

[0001] This application claims the benefit of United States Provisional Patent Application Serial No. 63 / 573,124, filed April 2, 2024, which is incorporated herein by reference in its entirety.

[0004] BACKGROUND

[0005]

[0002] Proteomics involves a variety of technologies, aimed at studying the identity and quantity of proteins that are expressed in cells, on cells, excreted by cells as well as their interactions with other cellular components. The ability to understand how proteins work and interact in cells is a critical component in studying carcinogenesis and diseases states, and how to potentially treat cancers and diseases by discovering biomarkers that are associated with such states. As such, a better understanding of proteins and how they work, or do not work, has application in screening, diagnosing, staging, prognosis, treatment regimen success, and failure for diseases and cancer.

[0006] SUMMARY

[0007]

[0003] There is a need for assays that are capable of assaying for multiple different targets, for example targets associated with immune response activation and proliferation, to advance our understanding of this important body defense system and potential modulation potential in fighting disease. The present disclosure relates to methods, compositions and systems for targeted detection of proteins. Immune system proteins are used in this disclosure to demonstrate that the methods disclosed herein are useful in protein identification. Immune system proteins are critical components of a healthy immune system. It is important to have mechanisms to study their characteristics when they are healthy and performing as expected and also when they malfunction. Equally important is determining patterns of immune cells in diseased and cancerous tissues. The methods, compositions and systems described herein provide a way to perform immune system related protein profiling to better understand the nexus between our immune system and disease states.

[0008]

[0004] Aspects disclosed herein provide methods for identifying a presence of a protein of interest from a sample, comprising: a) binding an antibody-oligonucleotide conjugate to the protein of interest from the sample, wherein the antibody-oligonucleotide conjugate comprises an antibody that recognizes and binds to the protein of interest and an oligonucleotide that is attached to the antibody, wherein the oligonucleotide comprises at least two functional sequences and at least one barcode that identifies the antibody; b) hybridizing the oligonucleotide of the antibody-oligonucleotide conjugate to a recognition element, wherein the recognition element comprises a 5’ end and a 3’ end that are complementary to the at least one barcode of the oligonucleotide of the antibody-oligonucleotide conjugate, and a code that uniquely identifies the recognition element that is hybridized to the oligonucleotide of the antibody-oligonucleotide conjugate; c) ligating the 5’ end and the 3’ end of the recognition element that is hybridized to the oligonucleotide of the antibody-oligonucleotide conjugate, thereby generating a ligated recognition element; d) amplifying the ligated recognition element to generate a concatemeric amplification product; and e) determining a sequence of the code in the concatemeric amplification product, and using the sequence of the code to correlate the code with the presence of the protein of interest from the sample. In some embodiments, the method further comprises amplifying the oligonucleotide of the antibody-oligonucleotide conjugate bound to the protein of interest, thereby generating a barcoded amplicon. In some embodiments, the methods further comprises hybridizing the barcoded amplicon to the recognition element. In some embodiments, a plurality of antibody-oligonucleotide conjugates are hybridized to a plurality of proteins of interest from the sample, and wherein a plurality of recognition elements are hybridized to the plurality of oligonucleotides of the plurality of antibody-oligonucleotide conjugates, for determining the presence of the plurality of proteins of interest from the sample. In some embodiments, the methods further comprises amplifying the plurality of oligonucleotides of the plurality of antibody-oligonucleotide conjugates bound to the plurality of proteins of interest, thereby generating a plurality of barcoded amplicons. In some embodiments, the methods further comprises, hybridizing the plurality of barcoded amplicons to the plurality of recognition elements. In some embodiments, the sample comprises a blood sample, a tissue sample, a cell culture sample, a biopsy sample, or a buffy coat sample. In some embodiments, the sample comprises a lysate or a permeabilized sample. In some embodiments, the protein of interest is associated with one or more immune cells comprising one or more of a T lymphocyte, a B lymphocyte, or a natural killer lymphocyte, and wherein the one or more of the T lymphocyte, the B lymphocyte, or the natural killer lymphocyte is present in the sample. In some embodiments, the one or more immune cells comprises the T lymphocyte. In some embodiments, the T lymphocyte is selected from the group consisting of CD3, CD4, CD8, CD25, CTLA-4, IFNg, IL-10, IL-2, IL-6, LAG-3, PD-1, PD-L1, and TNFa. In some embodiments, the plurality of the antibody-oligonucleotide conjugates are bound to proteins on an immune cell, wherein the proteins on the immune cell comprise two or more of CD3, CD4, CD8, CD25, CTLA-4, IFNg, IL-10, IL-2, IL-6, LAG-3, PD-1, PD-L1, or TNFa. In some embodiments, the method is performed concurrently for two or more of CD3, CD4, CD8, CD25, CTLA-4, IFNg, IL- 10, IL-2, IL-6, LAG-3, PD-1, PD-L1, or TNFa. In some embodiments, the method is performed concurrently for IFNg, IL- 10, IL-2, IL-6, LAG-3, PD-1, PD-L1, or TNFa. In some embodiments, the method is performed concurrently for CD3, CD4, CD8, CD25, CTLA-4, LAG-3, PD-1, or PD-L1. In some embodiments, the at least two functional sequences of the oligonucleotide are selected from the group consisting of a cleavage site sequence, an amplification primer binding site sequence, and a capture sequence. In some embodiments, the at least two functional sequences of the oligonucleotide comprise amplification primer binding sites for generating barcoded amplicons. In some embodiments, the oligonucleotide comprises one barcode. In some embodiments, the oligonucleotide comprises two barcodes. In some embodiments, the at least two functional sequences of the oligonucleotide flank the at least one barcode. In some embodiments, the amplifying the oligonucleotide of the antibody - oligonucleotide conjugate comprises polymerase chain reaction. In some embodiments, the amplifying the ligated recognition element comprises rolling circle amplification or multiple strand displacement amplification. In some embodiments, the code comprises one or more nucleic acid segments. In some embodiments, the determining the sequence of the code comprises: (i) hybridizing one or more detection polynucleotides to the one or more nucleic acid segments, or portions thereof, of the code;(ii) imaging the hybridizing of the one or more detection polynucleotides to the one or more nucleic acid segments; (iii) generating one or more signals from the imaging; and (iv) decoding the one or more signals. In some embodiments, the determining the sequence of the code comprises:(i) hybridizing a plurality of detection polynucleotides to the one or more nucleic acid segments, or portions thereof, of the code; (ii) imaging the of the plurality of detection polynucleotides to the one or more nucleic acid segments; (iii) generating a plurality of signals from the imaging; and (iv) decoding the plurality of signals. In some embodiments, the decoding the one or more signals or the decoding the plurality of signals comprises soft decision decoding. In some embodiments, the one or more detection polynucleotides or the plurality of detection polynucleotides comprise at least two different fluorescent moieties.

[0009]

[0005] Aspects disclosed herein provide compositions, comprising: a) a recognition element, wherein the recognition element comprises: i) a 5’ end and a 3’ end adjacently hybridized to an oligonucleotide of an antibody-oligonucleotide conjugate, wherein the oligonucleotide comprises a barcode that identifies a protein that is recognized and bound by the antibody of the antibody-oligonucleotide conjugate; and ii) a code comprising one or more nucleic acid segments, wherein the code uniquely identifies the recognition element, wherein the 5’ end and the 3’ end are ligated together to generate a circularized, ligated recognition element. In some embodiments, the composition further comprises a ligase. In some embodiments, the composition further comprises an exonuclease.

[0006] Aspects disclosed herein provide methods for identifying a presence of a protein of interest in a sample, comprising: a) binding an antibody-oligonucleotide conjugate to the protein of interest in the sample, wherein the antibody-oligonucleotide conjugate comprises an antibody that recognizes and binds to the protein of interest and an oligonucleotide that is attached to the antibody, wherein the oligonucleotide comprises at least two functional sequences and one or more barcodes that identify the antibody; b) amplifying the oligonucleotide of the antibody- oligonucleotide conjugate, or a portion thereof, thereby generating a plurality of barcoded amplicons from the oligonucleotide of the antibody-oligonucleotide conjugate; c) hybridizing a barcoded amplicon of the plurality of barcoded amplicons to a recognition element, wherein the recognition element comprises a 5’ end and a 3’ end that are complementary to one or more barcodes of a barcoded amplicon of the plurality of barcoded amplicons, and a code that uniquely identifies the recognition element hybridized to the barcoded amplicon of the plurality of barcoded amplicons; d) ligating the 5’ end and 3’ end of the recognition element hybridized to the barcoded amplicon of the plurality of barcoded amplicons, thereby generating a ligated recognition element; e) amplifying the ligated recognition element to generate a concatemeric amplification product; and f) determining a sequence of the code in the concatemeric amplification product, and using the sequence of the code to correlate the code with the protein of interest in the sample. In some embodiments, a plurality of antibody-oligonucleotide conjugates are hybridized to a plurality of proteins of interest from the sample, and wherein a plurality of recognition elements are hybridized to the plurality of barcoded amplicons, for determining the presence of a plurality of proteins of interest from the sample. In some embodiments, the protein of interest is selected from the group consisting of CD3, CD4, CD8, CD25, CTLA-4, IFNg, IL-10, IL-2, IL-6, LAG-3, PD-1, PD-L1, and TNFa. In some embodiments, the protein of interest comprises two or more of CD3, CD4, CD8, CD25, CTLA- 4, IFNg, IL- 10, IL-2, IL-6, LAG-3, PD-1, PD-L1, and TNFa. In some embodiments, the method is performed concurrently for two or more of CD3, CD4, CD8, CD25, CTLA-4, IFNg, IL-10, IL-2, IL-6, LAG-3, PD-1, PD-L1, or TNFa. In some embodiments, the method is performed concurrently for IFNg, IL- 10, IL-2, IL-6, LAG-3, PD-1, PD-L1, or TNFa. In some embodiments, the method is performed concurrently for CD3, CD4, CD8, CD25, CTLA-4, LAG-3, PD-1, or PD-L1. In some embodiments, the at least two functional sequences of the oligonucleotide of the antibody-oligonucleotide conjugate are selected from the group consisting of a cleavage site sequence, an amplification primer binding site sequence, and a capture sequence. In some embodiments, the at least two functional sequences of the oligonucleotide comprise amplification primer binding sites for generating the plurality of barcoded amplicons. In some embodiments, the oligonucleotide comprises one barcode. In some embodiments, the oligonucleotide comprises two consecutive barcodes. In some embodiments, the at least two functional sequences of the oligonucleotide flank the one or more barcodes. In some embodiments, the amplifying the oligonucleotide of the antibody-oligonucleotide conjugate comprises polymerase chain reaction. In some embodiments, the amplifying the ligated recognition element comprises rolling circle amplification or multiple strand displacement amplification. In some embodiments, the code comprises one or more nucleic acid segments. In some embodiments, the determining the sequence of the code comprises: (i) hybridizing one or more detection polynucleotides to the one or more nucleic acid segments, or portions thereof, of the code;(ii) imaging the hybridizing of the one or more detection polynucleotides to the one or more nucleic acid segments;(iii) generating one or more signals from the imaging the hybridizing; and (iv) decoding the one or more signals. In some embodiments, the decoding the one or more signals comprises soft decision decoding. In some embodiments, the determining the sequence of the code comprises next generation sequencing. In some embodiments, the one or more detection polynucleotides comprises a fluorescent moiety. In some embodiments, the fluorescent moiety is different for two or more of the one or more detection polynucleotides.

[0007] Aspects disclosed herein provide methods for identifying a presence of an immune system protein in a sample, comprising: a) binding an antibody-oligonucleotide conjugate to an immune system protein from the sample, wherein the antibody-oligonucleotide conjugate comprises an antibody that recognizes and binds to the immune system protein and an oligonucleotide that is attached to the antibody, wherein the oligonucleotide comprises one or more barcodes that identify the antibody; b) hybridizing the oligonucleotide of the antibody- oligonucleotide conjugate to a recognition element, wherein the recognition element comprises a 5’ end and a 3’ end that are complementary to the one or more barcodes of the oligonucleotide, and a code that uniquely identifies the recognition element that is hybridized to the oligonucleotide of the antibody-oligonucleotide conjugate; c) ligating the 5’ end and 3’ end of the recognition element hybridized to the oligonucleotide of the antibody-oligonucleotide conjugate, thereby generating a ligated recognition element; d) amplifying the ligated recognition element to generate a concatemeric amplification product; and e) determining a sequence of the code in the concatemeric amplification product, and using the sequence of the code to correlate the code with the presence of the immune system protein. In some embodiments, the methods further comprises amplifying the oligonucleotide of the antibody- oligonucleotide conjugate, thereby generating a barcoded amplicon. In some embodiments, the methods further comprises hybridizing the barcoded amplicon to the recognition element. In some embodiments, a plurality of antibody-oligonucleotides are hybridized to a plurality of immune system proteins from the sample, and wherein a plurality of recognition elements are hybridized to a plurality of oligonucleotides of the plurality of antibody-oligonucleotide conjugates, for determining the presence of the plurality of proteins from the sample. In some embodiments, the methods further comprises amplifying the plurality of oligonucleotides of the plurality of antibody-oligonucleotide conjugates, thereby generating a plurality of barcoded amplicons. In some embodiments, the methods further comprises hybridizing the plurality of barcoded amplicons to the plurality of recognition elements. In some embodiments, the sample comprises a blood sample, a tissue sample, a cell culture sample, a biopsy sample, or a buffy coat sample. In some embodiments, the sample comprises a lysate or a permeabilized sample. In some embodiments, the immune system protein is associated with one or more immune cells comprising one or more of a T lymphocyte, a B lymphocyte, or a natural killer lymphocyte. In some embodiments, the one or more immune cells comprises the T lymphocyte. In some embodiments, the T lymphocyte is selected from the group consisting of CD3, CD4, CD8, CD25, CTLA-4, IFNg, IL-10, IL-2, IL-6, LAG-3, PD-1, PD-L1, and TNFa. In some embodiments, the T lymphocyte comprises two or more of CD3, CD4, CD8, CD25, CTLA-4, IFNg, IL-10, IL-2, IL-6, LAG-3, PD-1, PD-L1, or TNFa. In some embodiments, the method is performed concurrently for two or more of CD3, CD4, CD8, CD25, CTLA-4, IFNg, IL-10, IL-2, IL-6, LAG-3, PD-1, PD-L1, or TNFa. In some embodiments, the method is performed concurrently for IFNg, IL- 10, IL-2, IL-6, LAG-3, PD-1, PD-L1, or TNFa. In some embodiments, the method is performed concurrently for CD3, CD4, CD8, CD25, CTLA-4, LAG-3, PD-1, or PD-L1. In some embodiments, the oligonucleotide of the antibody- oligonucleotide conjugate further comprises at least two functional sequences selected from the group consisting of a cleavage site sequence, an amplification primer binding site sequence, and a capture sequence. In some embodiments, the at least two functional sequences comprise amplification primer binding sites for generating the barcoded amplicons. In some embodiments, the oligonucleotide of the antibody-oligonucleotide conjugate comprises one barcode. In some embodiments, the oligonucleotide of the antibody-oligonucleotide conjugate comprises two barcodes. In some embodiments, the at least two functional sequences of the oligonucleotide flank the one or more barcodes. In some embodiments, amplifying the ligated recognition element comprises rolling circle amplification or multiple strand displacement amplification. In some embodiments, the code comprises one or more nucleic acid segments. In some embodiments, the determining the sequence of the code comprises hybridizing one or more detection polynucleotides to the one or more nucleic acid segments of the code to produce one or more signals, and decoding the one or more signals. In some embodiments, the decoding the one or more signals comprises soft decision decoding. In some embodiments, the one or more detection polynucleotides comprise a fluorescent moiety. In some embodiments, the methods further comprises imaging the fluorescent moiety of the hybridized detection polynucleotide, wherein the hybridizing of the one or more detection polynucleotides and the imaging is done for one or more cycles to generate a plurality of images. In some embodiments, the plurality of images are combined and used as a recognition element profile. In some embodiments, the methods further comprises soft decision decoding the recognition element profile.

[0010]

[0008] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative examples of the present disclosure are shown and disclosed. As will be realized, the present disclosure is capable of other and different examples, such as other protein types that are not immune cell related, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0011] INCORPORATION BY REFERENCE

[0012]

[0009] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014]

[0010] The features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present methods and systems will be obtained by reference to the following detailed description that sets forth illustrative examples, in which the principles of the methods and systems are utilized, and the accompanying drawings of which:

[0015] [OH] Fig. 1 is an example of a recognition element used in methods of the present disclosure.

[0012] Fig. 2 is an example of a workflow for generating concatemeric amplification products used in methods of the present disclosure.

[0016]

[0013] Fig. 3A provides an example of a detection polynucleotide used in methods of the present disclosure.

[0017]

[0014] Fig. 3B provides an example of a detection polynucleotide hybridized to an amplification product.

[0015] Fig. 4 is a schematic diagram of an example of a soft decision decoding pipeline for decoding a code of a recognition element.

[0018]

[0016] Fig. 5 is an example instrument that comprises a computer system for use with the methods described herein.

[0019]

[0017] Fig. 6 is an example of an application provision system for use with the methods described herein.

[0020]

[0018] Fig. 7 is an example of an application provision system for use with the methods described herein.

[0021]

[0019] Fig. 8 is an example of an antibody-oligonucleotide conjugate useful in the disclosed methods.

[0022]

[0020] Fig. 9 is an example of a recognition element that recognizes and interacts with the oligonucleotide of an antibody-oligonucleotide conjugate. This embodiment shows two barcodes present on the oligonucleotide.

[0023]

[0021] Fig. 10 is an example of a workflow of the present disclosure for protein detection and identification using the antibody-oligonucleotide conjugate of Fig. 8.

[0024]

[0022] Fig. 11 is an example of a standard curve for protein quantitation.

[0025]

[0023] Fig. 12 is an example of a graph demonstrating that the workflow of the present disclosure is able to successfully generate concatemers of targeted recognition elements which are used to identify specific T cell protein targets.

[0026]

[0024] Fig. 13A is an example of a graph showing the lower limit of detection, LLoD for protein target detection and linear range of detection.

[0027]

[0025] Fig. 13B is an example of a graph showing the lower limit of detection, LLoD for protein target detection and the lower limit of detection expanded from Fig. 13A.

[0028]

[0026] Fig. 14A shows example results of the effect of oligonucleotide conjugation to an antibody for oligonucleotide detection and comparison to synthetic free barcoded oligonucleotide.

[0029]

[0027] Fig. 14B shows example results of the effect of oligonucleotide conjugation to an antibody for oligonucleotide detection and comparison to background matrix.

[0030]

[0028] Fig. 15 shows a workflow for protein detection in and on peripheral blood mononuclear cells (PBMCs).

[0031]

[0029] Fig. 16A shows results of performing the workflow illustrated in Fig. 15, including detection and quantitation of surface and intracellular proteins from a single PMBC.

[0032]

[0030] Fig. 16B shows results of performing the workflow illustrated in Fig. 15, including the responsiveness of the assay when PMBCs are stimulated prior to assay. DETAILED DESCRIPTION

[0033]

[0031] A body's immune system comprises an arsenal of proteins for use in fighting off pathogens, infections, diseases, and cancers. Immune cells, and proteins that work in combination with immune cells such as inflammation related proteins, are part of that response. Immune cells that help mediate an immune response include white blood cells or leukocytes, of which there are multiple kinds which can be deployed by the body depending on its needs for defense. Lymphocytes are one group of white blood cells which can be further subdivided into various types of lymphocytes such as T lymphocytes that arise from stem cells in the bone marrow and mature in the thymus (e.g., thymus lymphocytes or T cells). T cells function as part of a healthy immune system and are a critical tool of the adaptive immune system helping to safeguard the body against infection and malignancy. T cells and proteins related to T cell mediated immune response can attack an invading cell or virus, but a T cell can attack one type of cell or virus as such T cells, and proteins related to their mediated immune response, may be highly specific immune missiles that target specific antigens on a cell or virus.

[0034]

[0032] Due to immune cells and immune response related proteins and their ability to support the body in guarding against infections and diseases, it is important to study their ability to do so and the conditions where they are compromised. Molecular assays, such as immunoassays and nucleic acid assays, may be used to detect one or more targets from a sample.

[0035]

[0033] Proteins carry out numerous functions in a living system and when a protein is mutated or rendered ineffective, catastrophic effects may happen to the system. As such, having tools to study and understand the proteome in a system is of critical importance in understanding when a body is well, or not.

[0036]

[0034] The present disclosure provides methods for studying proteins in a cell, tissue, plasma, serum or other sample type. In this disclosure proteins found on, in or around immune cells were evaluated to demonstrate the methods of the present disclosure in studying proteins. Immune related proteins were chosen for evaluation in understanding infection, disease and cancer and how the body responds. While immune cells and the system as a whole is meant to eliminate pathological microbes and viruses, and indeed other cells that have unnatural proliferative abilities (e.g., cancer cells), it may at the same time manage excessive damage to non-diseased cells and non-pathogenic microbes. As such, immune system protein were used as targets of interest, however any proteins could equally be identified using the methods described herein.

[0035] T lymphocytes, or T cells, are immune system cells that are able to discern host cells from pathogenic microbes, viruses, parasites and other non-host invaders. T cells have evolved an elegant mechanism for recognizing foreign antigens together with host antigens. As such, the ability of T cells to attack and remove non-host invaders, while at the same time managing damage to host tissues, is critical.

[0037]

[0036] The present disclosure uses T cell related protein detection as a predicate set of proteins for demonstrating the utility of the methods, composition and systems disclosed herein because of their importance in the human body’s ability to fight off foreign invaders. There are many other proteins in cells that make up a biological system that could also be studied, and it is contemplated that the methods described herein are equally applicable to those cellular proteins, be they immune cells or not, including, but not limited to, proteins in immune cells such as B lymphocytes (B cells), natural killer lymphocytes (NK cells), natural killer T lymphocytes (NK- T cells), neutrophils, macrophages, monocytes, eosinophils, basophils, and mast cells, proteins in cancer cells, proteins in neurological cells, proteins in liver cells, proteins in kidney cells, proteins in lung cells, proteins in brain cells, proteins in skin cells and protein in the environments that surround these cell types. The methods of the present disclosure are not limited to identifying proteins in, or around immune cells and demonstrating their ability to detect proteins related to immune cell mediated immune response can be extrapolated and applied to other cells and environments in a biological system.

[0038]

[0037] With regards to studying the immune system, and understanding of one, many or all of the different immune cells and proteins related to immune cell mediated response can be used in determining treatments and therapies that may work in tandem with an immune response or diminish an immune response that has blown out of control, which can be personalized from one person to the next.

[0039]

[0038] Tools that can study immune cells in a system are needed to support this understanding. The present disclosure relates to methods, compositions, and systems for identification of immune cells in a sample.

[0040]

[0039] As used herein, “about” means approximately, roughly, around or in the region of. When used to describe a numerical range, it modifies that range by extending the boundaries above and below the numerical values, for example by 10% higher or lower than the stated numerical range.

[0041]

[0040] As used herein, “include”, “including” and the like are used in an inclusive manner as opposed to an exclusive or exhaustive manner. Such as “including but not limited to”. Include, including, and the like are considered open ended in their usage.

[0042]

[0041] As used herein, “linked”, “linking”, “bind”, “binding” can refer to interactions between two or more entities, such as nucleic acids, a nucleic acid and a protein, or a protein and a protein. Linking and binding events can be covalent or non-covalent in nature. For example, linking and binding encompass enzymatic or chemical interactions between two entities, which can be reversible or non-reversible. The interactions include any degree of hybridization between two nucleic acid sequences, for example hydrogen bonding, van der Waals interactions and other weak interactions between nucleic acid molecules. Additionally, linked and linking can further include the use of a “linker,” which attach one entity to another. Linkers can be amino acid sequences, nucleic acid sequences, chemistries, or a combination thereof.

[0043]

[0042] The terms “decoding” with respect to a code includes determining the presence of a code or the probability of the presence of a code. The code can be detected in a number of ways, for example by determining the codes using next generation sequencing chemistries to determine the sequence of the code in a base by base fashion, or by using detection oligonucleotide complexes that comprise a detectable moiety such as a fluorescent moiety. The data generated from detecting the code can be “decoded” by applying hard or soft decision detecting algorithms which is then used to identify the presence of the code and, by proxy, the presence or absence of the target molecule from the sample.

[0044]

[0043] As used herein, the terms “target” or “target molecule can be any molecule that is detected directly or indirectly. Non-limiting examples of a target molecule include a protein, peptide, and nucleic acid. In some embodiments, the target molecule is a target nucleic acid molecule, the sequence of which is entirely or partially known. In some embodiments, the target molecule is a protein detected using an antibody-oligonucleotide conjugate, wherein the antibody-oligonucleotide conjugate may comprise an oligonucleotide that is or comprises a target nucleic acid molecule. In either embodiment, the target nucleic acid molecule may have a nucleic acid sequence that is detected directly using the methods, systems and kits of the present disclosure.

[0045]

[0044] As used herein, the term “target sequence of interest” or “target sequence” is a nucleic acid sequence of a target nucleic acid molecule.

[0046]

[0045] As used herein, a “sample” comprising a target molecule can be from any species, human or non-human primate, mammals, aves, bacteria, viral, etc. The present disclosure is not limited by the source of the sample. Examples of samples include biological samples, such as tissues, cells, whole blood, lymphatic fluid, serum, plasma, sweat, tear, saliva, sputum, cerebrospinal fluid, amniotic fluid, seminal fluid, vaginal excretion, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, transudates, exudates, cystic fluid, bile, urine, gastric fluid, intestinal fluid, fecal samples, liquids containing single or multiple cells, liquids containing organelles, fluidized tissues, fluidized organisms, liquids containing multi-celled organisms, biological swabs and biological washes. Samples may be from any organism (e.g., prokaryotes, eukaryotes, plants, animals, humans) or other types of samples (e.g., environmental or forensic samples). Alternatively, a sample can be obtained through primary cell lines, or archived samples in the form of preserved samples such as formalin fixed paraffin embedded tissues, or fresh frozen tissue samples. In some embodiments, the sample comprises target molecules that have been extracted or isolated from a biological sample, such as target nucleic acids, using any technique that does not interfere with subsequent analysis. In some embodiments, the sample comprises target molecules that are proteins and are surface proteins, intracellular proteins, transmembrane proteins, or extracellular proteins.

[0047]

[0046] Disclosed herein, in some embodiments, are methods and compositions for determining a target molecule in an assay. In some embodiments, the assay is a solution-based assay. In some embodiments, the assay is a surface-bound assay. In some embodiments, the assay is a hybrid assay that includes a surface-bound component and a solution-based component. In some embodiments, the assay is performed in tubes or in a plate-based format, such as a multi-well plate for example a 24 well, 48 well, or 96 well plate. In some embodiments, a multi-well plate may include, for example, an array of nanowells. In some embodiments, the assay may be performed on a microfluidics device. In some embodiments, the assay is performed partially in tubes and partially in a multi-well plate.

[0048]

[0047] In some embodiments, a recognition element is used in the assay. A recognition element used in the assay comprises sequences that are complementary to a target sequence of interest at the 5’ and 3’ ends of the recognition element, wherein the complementary sequence can hybridize a target sequence of interest, a code sequence that can be used to identify the target sequence of interest that has hybridized to its complement on the recognition element, and one or more functional sequences such as sequencing primer binding sites, one or more amplification primer binding sites, unique molecular identifier sequences, sample indexes, or combinations thereof. In some embodiments, an amplification primer binding site may be adjacent to the code in a recognition element. The amplification primer binding site(s) may, in some cases, be universal primer sequence(s) that are common to all recognition elements in a set of recognition elements. Amplification primer binding site sequences may also be a code sequence or a portion thereof. A code sequence can be a combination of a number of subsequences, called nucleic acid segments, wherein their combination can identify a target sequence of interest that has hybridized to a recognition element. In some embodiments, an amplification primer can be a nucleic acid segment or a portion thereof. Unique identifier sequences and sample indexes, which oftentimes find utility in next generation sequencing reactions for counting, error correction and sample identification purposes, may also be part of code such as one or more nucleic acid segments.

[0049]

[0048] In some embodiments, once a recognition element has recognized and hybridized to its target of interest, the recognition element is circularized and ligated to generate a circular recognition element. The circular recognition element can be amplified in anticipation of a decoding event to identify the code of the recognition element associated with the original target of interest that hybridized to the recognition element. Amplification may be by any method of amplification, including for example, nucleic acid extension, PCR, isothermal amplification, rolling circle amplification (RCA), multiple strand displacement amplification, and / or ultrarapid amplification. Surface based amplification may be performed using PCR with surface-anchored primers (e.g., bridge amplification technology), or recombinase polymerase amplification (RPA) (e.g., ExAmp technology).

[0050]

[0049] In one embodiment, the amplification operation comprises a RCA reaction to generate a concatemeric amplification product.

[0051]

[0050] In one embodiment, a recognition element may include a sequence which may prevent RCA of the recognition element while allowing for linear double-stranded PCR products. The non-extendable sequence may, for example, be located between a pair of amplification primer binding site sequences present on the recognition element. In one embodiment, a recognition element may include a restriction enzyme site that may be cleaved to yield a linear DNA molecule.

[0052]

[0051] In some embodiments, an amplification product may be sequenced to determine the nucleotide sequence of the code associated with the target molecule of interest. Any sequencing technology may be used to sequence the product. Examples of sequencing technologies that may be used include sequencing by synthesis, avidity sequencing, sequencing by hybridization, sequence by transient binding, sequencing by ligation, and nanopore sequencing.

[0053]

[0052] In some embodiments, a sequencing library may be generated from a set of recognition elements or complements or amplicons thereof. The library may be sequenced to determine the code of the recognition element associated with a target molecule of interest. The code sequence may then be used as a digital count of the target molecule specific decoding event. In one embodiment, a sequencing library may be generated from a circularized recognition element. In another embodiment, a sequencing library may be generated from an amplification product of a recognition element. In one embodiment, an amplification product or a portion thereof that includes at least the code may be directly sequenced to determine the code associated with the target molecule of interest.

[0054]

[0053] Fig. 2 is an example of an encoded assay for use with the detection polynucleotides disclosed herein. Referring to the example in Fig. 2, a linear recognition element 210 comprises a 5' end 220a which is complementary to a portion of a target nucleic acid interest 222, a 3' end 220b which is complementary to another portion of a target nucleic acid of interest 222 from a sample, a code 216, and additional functional sequences 212, 214, 218 if desired such as amplification primer binding sites, capture sequencing, cleavage sites, sequencing primer binding sites, unique molecular identifiers, and the like. A target nucleic acid of interest 222 which is complementary to the 5' 220a and 3' 220b ends of the linear recognition element 210 hybridizes to the linear recognition element, thereby bringing the ends in proximity for ligating to generate a circular and ligated recognition element 225. The circular and ligated recognition element 225 is subjected to extension amplification using one of the functional sequences 212, 214, 218, or even the code 216 or a portion thereof, as a primer binding site. The result is a concatemeric amplification product 230 which can be decoded using the detection polynucleotides disclosed herein for identifying and determining the presence of the target nucleic acid of interest from a sample.

[0055]

[0054] Additional examples of encoded assays can be found in WO2022 / 109496A2, which is incorporated herein by reference in its entirety.

[0056]

[0055] The methods and compositions described herein include providing recognition elements to an encoded assay for identifying the presence of a target molecule of interest from a sample. In some embodiments, a plurality of recognition elements is provided. In some embodiments, each recognition element in the plurality of recognition elements comprises one or more target recognition regions. The target recognition regions of the recognition elements comprise one or more nucleic acid sequence(s) configured to hybridize to a target nucleic acid molecule. In some embodiments, the one or more nucleic acid sequences hybridize to one or more target nucleic acid sequences of the target nucleic acid molecule. In some embodiments, the target recognition region is configured to hybridize to one or more regions of the target nucleic acid molecule flanking a target of interest (e.g., SNP, indel, and so on). In some embodiments, the target recognition region is configured to hybridize to a variant nucleic acid of interest (e.g., the target recognition region base pairs with the SNP when the target of interest is a SNP).

[0057]

[0056] As shown in Fig. 1, a non-limiting example of a recognition element used in encoded assays described herein comprises two target recognition regions, one at the 5’ end and another at the 3’ end. In some embodiments, a recognition element further comprises a code. In Fig. 1, the example code is made up of four nucleic acid segments. However, the number of nucleic acid segments, be it one or more than one, is not limiting and the number of segments depends on the complexity of the assay (e.g., how many targets of interest are to be identified). In some embodiments, a target nucleic acid molecule of interest itself comprises the code, or an additional code such as a barcode that identifies another target molecule of interest such as a protein. In either embodiment, the code may be detected as a proxy for the target molecule, be it a nucleic acid or a protein.

[0057] In some embodiments, the structure of the recognition element may vary. In some embodiments, the structure of the recognition element may configure into a specific structure when hybridized to a target nucleic acid. Non-limiting examples of a recognition element configuration may include a padlock probe, a molecular inversion probe, a hairpin oligonucleotide, a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a combination thereof. The recognition element configuration may include a padlock probe configuration. The recognition element configuration may include a molecular inversion probe configuration. In some embodiments, the recognition element is linear prior to hybridization to its complementary target nucleic acid of interest. In some embodiments, the linear recognition element is circularized once hybridized to the respective target nucleic acid of interest and ligated thereafter. In some embodiments, the recognition element is circular prior to hybridization to the target nucleic acid of interest. In one embodiment, the target nucleic acid of interest may serve as a primer for an extension reaction, for example to initiate rolling circular amplification of the ligated recognition element.

[0058]

[0058] In some embodiments, the recognition element is configured to be a padlock probe once the recognition element is hybridized to the target nucleic acid of interest. Padlock probes may be referred to as linear oligonucleotides whose ends are complementary to adjacent target sequences, or to non-adjacent target sequences thereby leaving a gap between the ends of the hybridized recognition element. Upon hybridization to a target nucleic acid, the two ends (e.g., 5’ end and 3’ end) of the recognition element are adjacently located, generating a padlock probe configuration for subsequent ligation. Alternatively, the two ends of the recognition element are brought in proximity to, but not directly adjacent to, each other upon hybridization to a target nucleic acid. In this instance, a gap is left between the 5' and 3' hybridized ends of the recognition element which can be filled in several ways, for example by extension of the 5' end until it is adjacent to the 3' end, or by hybridizing a third oligonucleotide that fills the gap. In any scenario, the recognition element ends are ligated together if hybridization, or hybridization and gap fill, occurs thereby generating circular and ligated recognition elements that are indicative of the hybridization event.

[0059]

[0059] In some embodiments, a recognition element further comprises one or more functional sequences. Functional sequences include, but are not limited to, primer binding sites, cleavage sites, unique molecular identifiers, capture sequences, or combinations thereof. The functional sequences may comprise primer binding sites. The functional sequences may comprise cleavage sites. The functional sequences may comprise unique molecular identifiers. The functional sequences may comprise capture sequences. In some embodiments, a primer binding site and / or a cleavage site are universal in nature, such that a plurality of recognition elements shares the same sequence(s). A unique molecular identifier could be included in a recognition element to identify a source of material, for error correction, as known in the art.

[0060]

[0060] The methods described herein relate to the use of a code for identifying a target nucleic acid of interest from a sample that hybridized to a recognition element to initiate a ligation event. A code in a recognition element may be used to associate the recognition element 5' and 3' end regions with a target nucleic acid of interest, thereby determining the presence of a target nucleic acid of interest from a sample without having to directly assay the target molecule itself. As such, a code in a recognition element may uniquely identify the presence of a target molecule from a sample. Using codes, any number of recognition elements can be multiplexed in one encoded assay as each code is unique and correlates to the presence of one target molecule. In some embodiments, the code is selected from a set of codes wherein the set of codes make up a “code space”. In some embodiments, the code comprises a plurality of nucleic acid segments, where each nucleic acid segment corresponds to one or more computational symbols, or colors, that are used in a detection and decoding process. Fig. 1 shows a non-limiting example where four nucleic acid segments make up the code of the recognition element, wherein each of the nucleic acid segments can be detected using detection polynucleotides as disclosed herein and the combination of the detected nucleic acid segments thereby builds a code profile which can be decoded and is unique to the target nucleic acid of interest from a sample. In some embodiments, the codes are detected as proxies, thereby serving as an indirect analysis of the presence of a target molecule from a sample as the code correlates with the presence of the target molecule that hybridized to the recognition element allowing ligation, amplification, detection and decoding. In some embodiments, if there is no hybridization of a target of interest to its complementary sequences of a recognition element, there is expected to be no ligation (e.g., as the 5’ and the 3’ ends of the recognition are not expected to be adjacent), no amplification and subsequently nothing to detect and decode. As such, in some embodiments, if there is no amplification product to detect, that would be an indication that the target molecule of interest was potentially absent from the sample, or at such a low incidence that hybridization resulted in too few amplification products to cross the threshold for detection and decoding.

[0061]

[0061] In some embodiments, each code from the set of codes is from a predetermined set of codes. In some embodiments, each code from the set of codes may be selected to ensure that the selected code differs from other codes in the set of codes. As such, in some embodiments, several selection criteria may be implemented to generate a set of codes, wherein each code of a set of codes comprises from one to more than one nucleic acid segment. In some embodiments, selection of the codes, or nucleic acid segments that make up a code, may incorporate a Hamming distance criterion.

[0062] In some embodiments, to generate a code selected from a set of codes for use in a recognition element, a Hamming distance (HD) selection criterion may be implemented between any two codes of the set of codes, and also between any two nucleic acid segments that may be used in a code. A Hamming distance between two codes in a set of codes may refer to the number of symbols, or nucleotides, that differ between the two codes in the set of codes. In essence, the Hamming distance measures the number of changes that would need to be made to a first code sequence to change the string of symbols, in this case nucleotides, to the second code. As such, a Hamming distance criterion used to select a code cannot be greater than the length of the code. For example, if the length of a code is measured by the number of cycles or flows of decoding runs or queries and that number being eight cycles, and if each cycle corresponds to one symbol or color, therefore eight symbols or colors, then the maximum Hamming distance is eight. In some embodiments, the Hamming distance may be a minimum Hamming distance. In some embodiments, the Hamming distance may be a maximum Hamming distance. In some embodiments, a minimum Hamming distance may be from about 2-10. In some embodiments, the Hamming distance is between 2-7. In some embodiments, the Hamming distance is between 3-5. The Hamming distance increases as the number of codes that can be used decreases, as the purpose of the code is to impart a way to uniquely identify one target molecule from another target molecule.

[0062]

[0063] The code may have a certain length in nucleotides. In some embodiments, the code has a length of greater than or equal to about three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides. In some embodiments, the code has a length of fewer than or equal to about 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 contiguous nucleotides. In some embodiments, the length is about 5 to 200, 10 to 150, 15 to 100, 20 to 90, or 30 to 80 contiguous nucleotides. The length of the code may be further divided into a number of discrete nucleic acid segments, as illustrated in Fig. 1.

[0063]

[0064] In some embodiments, each code from a set of codes is generated using a 4-ary nucleotide alphabet of A, C, G, and T. In some embodiments, each code of a set of codes is generated using a 3 -ary nucleotide alphabet of a set of three of A, C, G, and T. In some embodiments, the codes can be generated from arbitrary symbols, or colors, 1 to 4, corresponding to the fluorophores that are associated with a unique string of nucleotides. The numbers then can be used in the abstract but serve as a means to numerate colors or mixes of colors that are utilized to query the codes for decoding.

[0064]

[0065] In one embodiment, codes from one or more recognition elements are amplified, subjected to sequencing library preparation, and further amplified via bridge amplification to produce clusters of oligonucleotides comprising a code on a substrate for next generation sequencing. In one embodiment, codes from one or more recognition elements are amplified and attached to a substrate for immobilization, for example a bead to produce bead-attached oligonucleotides comprising a code. In one embodiment, codes are detected using a patterned array, such as a microarray comprising oligonucleotides which are complimentary to the codes, such that nucleic acids, for example recognition elements or amplicons, comprising a code are captured on a microarray for detection and decoding. In one embodiment, detection and decoding of the codes of a recognition element is done in situ, for example in a cell or tissue, after one or more recognition elements or amplicons or fragments thereof is applied to a cell or tissue for determining the presence of a target molecule in situ. In one embodiment, codes of a circularized recognition element, or amplicons thereof, are detected using an electronic or electrical sensing mechanism.

[0065]

[0066] In some embodiments, detection of a code is performed using detection polynucleotides that are complementary to the code, or a portion of the code. For example, in some embodiments, if one code comprises one nucleic acid segment, then one detection polynucleotide that is complementary to that nucleic acid segment, or a portion thereof, is added to the amplification product, hybridized and detected. In some embodiments, if the label on the detection polynucleotide is a fluorescent moiety, then detection is via fluorescence. In some embodiments, if more than one nucleic acid segment, or a portion thereof, is used to generate a code then, for example, each segment can have a complementary detection polynucleotide which is sequentially or concurrently added to an amplification product, hybridized and detected. For each probe complementary to each nucleic acid segment, or a portion thereof, a different emitting fluorescent moiety can be attached to each different detection polynucleotide and fluorescence is detected using different emission filters. Alternatively, in some embodiments, not each detection polynucleotide needs a uniquely emitting fluorescent moiety, as patterns of detection of fewer unique fluorescent moieties can be generated and as previously defined identify the presence of a target molecule. As such, there are various ways for detecting codes and aligning them with the target molecule to which they are correlated.

[0066]

[0067] A code of a recognition element can comprise one or more nucleic acid segments. For example, as shown in Fig. 1, the example code comprises four nucleic acid segments.

[0067]

[0068] In some embodiments, the recognition elements provided herein comprise a code comprising one or more nucleic acid segments. The one or more nucleic acid segments, or the complements thereof, within the code are used as a proxy for detection of the target molecule recognized by the recognition element.

[0069] In some embodiments, the number of nucleic acid segments present in a code of a recognition element are considered in the design of the recognition element. The number of segments in a code helps to determine the nucleotide length of the recognition element. For example, a recognition element that includes a code comprising five segments may comprise a greater nucleotide length than a recognition element that includes a code of only two segments. A recognition element with a larger nucleotide length may run up against synthesis limits and is at a greater risk of synthesis errors. Alternatively, a recognition element with a smaller nucleotide length may avoid synthesis limits and risks in synthesis errors. A recognition element with a larger nucleotide length may include less space for other portions of the recognition element, such as the target recognition regions, functional sequences, universal sequences, etc.

[0070] In some embodiments, the code comprises 2 to 10 nucleic acid segments. In some embodiments, the code comprises 2 to 8 nucleic acid segments. In some embodiments, the code comprises 3 to 5 nucleic acid segments. In some embodiments, the code comprises at least 4 nucleic acid segments, at least 5 nucleic acid segments, at least 6 nucleic acid segments, at least 7 nucleic acid segments, at least 8 nucleic acid segments, at least 9 nucleic acid segments, at least 10 nucleic acid segments.

[0068]

[0071] In some embodiments, each nucleic acid segment may comprise a length in nucleotides. In some embodiments, each nucleic acid segment may comprise a length of about 10 to about 30 nucleotides. In some embodiments, each nucleic acid segment may comprise a length of about 10 to about 25 nucleotides. In some embodiments, each nucleic acid segment may comprise a length of about 15 to about 20 nucleotides. In some embodiments, each nucleic acid segment may comprise a length of about 2 or more nucleotides, about 4 or more nucleotides, about 6 or more nucleotides, about 8 or more nucleotides, about 10 or more nucleotides, about 12 or more nucleotides, about 14 or more nucleotides, about 16 or more nucleotides, about 18 or more nucleotides, about 20 or more nucleotides, or about 22 or more nucleotides. In some embodiments, the nucleic acid segments in a code are of the same length. In some embodiments, the nucleic acid segments in a code are not the same length.

[0069]

[0072] As with a code in a recognition element, a Hamming distance selection criterion may be implemented between any two nucleic acid segments of a code. A Hamming distance between two nucleic acid segments in a code refers to the number of symbols that differ between the segments. In essence, the Hamming distance measures the number of changes that would need to be made to a first nucleic acid segment to change the string of symbols, or nucleotides, to a second nucleic acid segment. In some embodiments, the Hamming distance may be a minimum Hamming distance. In some embodiments, the Hamming distance may be a maximum Hamming distance. In some embodiments, a minimum Hamming distance may be from about 2-20, about 3-19, about 4-18, about 5-17, about 6-16, about 7-15, about 8-14, about 9-13, or about 10-12. In some embodiments, a minimum Hamming distance may be greater than or equal to about 2, greater than or equal to about 3, greater than or equal to about 4, greater than or equal to about 5, greater than or equal to about 6, greater than or equal to about 7, greater than or equal to about 8, greater than or equal to about 9, greater than or equal to about 10, greater than or equal to about

[0070] 11, greater than or equal to about 12, greater than or equal to about 13, greater than or equal to about 14, greater than or equal to about 15, greater than or equal to about 16, greater than or equal to about 17, greater than or equal to about 18, greater than or equal to about 19, or greater than or equal to about 20.

[0071]

[0073] In some embodiments, a nucleic acid segment may comprise a universal primer binding site for amplification. For example, a segment may comprise an amplification primer binding site for performing rolling circle amplification (RCA) for generating a plurality of concatemeric amplification products.

[0072]

[0074] In some embodiments, a nucleotide or nucleic acid sequence of each segment corresponds to one or more computational symbols, such as a detection color, for performing a decoding process. For example, one or more nucleic acid segments of a code may be detected with a first pool of detection polynucleotide complexes to produce one or more detectable binding complexes, for example by using a fluorescent label. In some embodiments, the one or more detectable binding complexes, once imaged, produce one or more optical signals such as fluorescence in a particular wavelength. When all or substantially all segments of the code are detected by iteratively applying additional pools of detection polynucleotide complexes to the amplification products, a series of optical signals, or a code profile, are observed and collated.

[0075] The application of detection polynucleotides to amplification products for decoding can be called a “flow” or "cycle" or “query”, wherein a flow, cycle or query is the number of times a particular segment of an amplification product is queried, or the number of times a detection polynucleotide is flowed over an amplification product in order to detect a nucleic acid segment sequence. If a nucleic acid segment is present, a detection polynucleotide that comprises a sequence complementary to that nucleic acid segment will hybridize to its complementary nucleic acid segment, or portion thereof, and the attached detectable label is detected, for example imaged. In some embodiments, one or more optical signals observed from querying an amplification product with detection polynucleotide complexes translates to one or more computational symbols such that each optical signal can be imaged and used for decoding. In some embodiments, a plurality of nucleic acid segments on a recognition element may correspond to at least three computational symbols. In some embodiments, the optical signal may be a color or a non-color. In some embodiments, the optical signal may be a combination of colors (e.g., when the detection polynucleotide complex comprises a plurality of detectable labels). In some embodiments, the computational symbols or colors can be referred to as numbers (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.). In some embodiments, each detection polynucleotide complex comprises a detectable label, as such for example when four different fluorescent moieties are used as detectable labels there are four symbols, 1 to 4. However, the number of symbols can be larger depending on the combination of detectable labels with each unique detection polynucleotide complex. For example, for a set of 16 unique detection polynucleotide complexes wherein each has one of four fluorescent moieties there would be 16 computational symbols used for detecting and decoding if all 16 unique detection polynucleotide complexes are used to detect an amplification product. However, additional ways to increase the number of computational symbols for decoding include, but are not limited to, adding levels of identifiability associated with a particular detectable signal such as whether a detectable signal is brighter or dimmer compared to a normal level of signal, whether there is a combination of detectable colors that is used to identify a particular nucleotide. As such, the number of computational symbols that could be used is only limited by practicality for any given assay.

[0076] In some embodiments, the methods described herein may use a number of computational symbols. The number of computational symbols used in the methods and systems described herein may be considered in the design of the recognition elements. For example, in some embodiments, a detection scheme using a larger number of computational symbols may lead to a larger code space and a greater number of codes that may be generated, which may allow for a greater amount of information that may be detected thereby allowing for a higher degree of assay target molecule multiplexing. In some embodiments, a detection scheme using a smaller number of computational symbols may be limited in the amount of information that can be detected. In some embodiments, using a larger number of computational symbols may result in a faster detection process (less time to determine a target molecule compared to using a smaller number of computational symbols). In some embodiments, a detection scheme using a larger number of computational symbols may require greater instrument complexity, which may lead to potential drawbacks such as color crosstalk, wherein the computational symbols used in the detection scheme may become difficult to distinguish from other computational symbols. In some embodiments, a greater number of computational symbols may require that a more complex detection tool be used.

[0073]

[0077] In some embodiments, each nucleic acid segment corresponds to a combination of computational symbols. In some embodiments, each nucleic acid segment corresponds to one or more computational symbols, two or more computational symbols, three or more computational symbols, four or more computational symbols, five or more computational symbols, six or more computational symbols, seven or more computational symbols, eight or more computational symbols, nine or more computational symbols, or 10 or more computational symbols. In some embodiments, each segment may correspond to 10 or less computational symbols, nine or less computational symbols, eight or less computational symbols, seven or less computational symbols, six or less computational symbols, five or less computational symbols, four or less computational symbols, three or less computational symbols, or two or less computational symbols.

[0074]

[0078] The methods described herein include amplification of a circularized and ligated recognition element. In some embodiments, the circularized recognition element is ligated. In some embodiments, a target nucleic acid molecule is amplified. In some embodiments, the amplification is selective amplification. For example, in some embodiments, amplification is able to occur if a target recognition region of a recognition element recognizes and binds to a complementary target nucleic acid of interest. In some embodiments, amplification occurs if a primer is used that is complementary to one or more of a portion of a recognition element, a portion of a nucleic acid segment of a code, or another sequence in the recognition element that is complementary to a primer used for amplification. In some embodiments, the amplification is non- selective. For example, in some embodiments random ers can be used to prime amplification from a recognition element.

[0075]

[0079] In some embodiments, the methods described herein may include selectively amplifying a subset of nucleic acids. For example, in some embodiments, a subset of a plurality of recognition elements hybridized to a plurality of target nucleic acid molecules may be amplified. The subset may comprise a percentage of the total amount of recognition elements hybridized to target nucleic acid molecules as described herein. In some embodiments, the subset may include 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more of the total amount of recognition elements hybridized to target nucleic acid molecules. In some embodiments, the subset may include 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less of the total amount of recognition elements hybridized to target nucleic acid molecules.

[0076]

[0080] In some embodiments, the amplification may include rolling circle amplification (RCA). In some embodiments, the RCA may generate a concatemer as an amplification product, wherein the concatemer contains multiple copies of a circularized ligated recognition element, including associated codes, target recognition regions, and any other functional sequences that are included in the circularized and ligated recognition element. In some embodiments, RCA may be performed while the circularized and ligated recognition element is in solution. In some embodiments, RCA may be performed on a circularized recognition element while the circularized recognition element is immobilized, either reversibly or non-reversibly, on a substrate or surface. In some embodiments, the substrate or surface is a solid support and includes, but is not limited to, a bead, a flow cell, a microwell, a nanowell, a well, a slide. In some embodiments, the substrate is glass such as optical glass of imaging quality. In some embodiments, the substrate is plastic, polycarbonate, etc. In some embodiments, the substrate is positively charged or negatively charged. In some embodiments, the substrate is an anionic substrate. In some embodiments, the substrate is a cationic substrate. In some embodiments, the substrate comprises an immobilization composition, such as polyacrylamide, branched PEI, linear PEI, poly(P-aminoester) and poly(amidoamine), PEG, a gel, poly-L-lysine, silane, agarose, muscle mimetic catecholamine polymer, and the like. In some embodiments, the substrate has no charge. In some embodiments, the substrate has no immobilization composition. In some embodiments, a substrate comprises a cationic polymer coated surface. An RCA reaction may be performed in the presence of a cationic polymer coated surface, resulting in simultaneous immobilization and amplification of a ligated recognition element. In some embodiments, the ligated recognition elements are allowed to pre-bind to a substrate, for example a substrate coated with a compound for immobilizing nucleic acids, thereby immobilized the ligated recognition elements prior to RCA. RCA primers may be supplied in solution or bound to the cationic polymer-coated surface prior to, or concurrent with, performing the RCA reaction.

[0077]

[0081] In some embodiments, amplification may include on-surface polymerase chain reaction (PCR), isothermal amplification, RCA, ultrarapid amplification, or a combination thereof. In some embodiments, amplification may include polymerase chain reaction (PCR). In some embodiments, PCR is multiplexed PCR. The amplification methods disclosed herein may include isothermal amplification. Non-limiting examples of isothermal amplification include Nicking endonuclease amplification reaction (NEAR), Transcription mediated amplification (TMA), Loop-mediated isothermal amplification (LAMP), Helicase-dependent amplification (HD A), Nucleic Acid Sequence Based Amplification (NASBA), Strand displacement amplification (SDA), Multiple Displacement Amplification (MDA), Rolling Circle Amplification (RCA), bridge amplification, or Ramification (RAM) amplification method. In some embodiments, the amplification method is provided in Fakruddin M, Mannan KS, Chowdhury A, Mazumdar RM, Hossain MN, Islam S, Chowdhury MA. Nucleic acid amplification: Alternative methods of polymerase chain reaction. J Pharm Bioallied Sci. 2013 Oct;5(4):245-52, which is hereby incorporated by reference in its entirety.

[0078]

[0082] The methods described herein include introducing detection polynucleotides, or detection polynucleotide complexes, to amplified recognition elements. A detection probe may be a single stranded oligonucleotide, or it may be partially single stranded and partially double stranded as a detection polynucleotide. In some embodiments, a detection oligonucleotide or a detection polynucleotide comprises a detectable label.

[0079]

[0083] In some embodiments, the methods described herein may include introducing one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, 10 or more, 15 or more, 20 or more, 25 or more, 50 or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, or 1,000 or more detection polynucleotides or polynucleotide complexes (or single stranded oligonucleotides) to an amplification product. In some embodiments, the methods described herein may include introducing 1,000 or less, 900 or less, 800 or less, 700 or less, 600 or less, 500 or less, 400 or less, 300 or less, 200 or less, 100 or less, 50 or less, 25 or less, 20 or less, 15 or less, 10 or less, nine or less, eight or less, seven or less, six or less, five or less, four or less, three or less, or two or less detection polynucleotides to an amplification product.

[0080]

[0084] In some embodiments, a detection oligonucleotide comprises a detectable label (e.g., fluorescent molecule). In some embodiments, a detection oligonucleotide is a single stranded oligonucleotide with a portion that is complementary to a code, or a portion of a code, and a detectable label. In some embodiments, a detection polynucleotide complex comprises two oligonucleotides. Fig. 3A shows a non-limiting example of a structure of a detection polynucleotide 300 comprising two oligonucleotides. A first detection oligonucleotide 330 comprises a detectable label 340. A second anchor oligonucleotide 350 comprises a portion that is complementary to the first detection oligonucleotide 310 and a second portion 320 that is complementary to a code or a portion 360 (e.g., a nucleic acid segment of a code, or a portion thereof). The first detection oligonucleotide 330 hybridizes to the second anchor oligonucleotide 350, thereby generating a detection polynucleotide or detection polynucleotide complex. For detection, a portion of the second anchor oligonucleotide 360 hybridizes to its code complement 370 as seen in Fig. 3B, and a signal is detected from the detectable label, thereby identifying the presence of a code which in turn is correlated back to the presence of a target nucleic acid of interest from a sample.

[0081]

[0085] In some embodiments, the detection polynucleotide may comprise various nucleotide lengths. In some embodiments, the detection polynucleotide may comprise a length of 5 to 25 nucleotides. In some embodiments, the detection polynucleotide may comprise a length of 5 to 20 nucleotides. In some embodiments, the detection polynucleotide may comprise a length of 5 to 15 nucleotides. In some embodiments, the detection polynucleotide may comprise a length of 5 to 10 nucleotides. In some embodiments, the detection polynucleotide may comprise a length of 5 to 8 nucleotides.

[0082]

[0086] In some embodiments, the detection oligonucleotide may comprise a length of between about 5 to about 100 nucleotides, between about 10 to about 80 nucleotides, between about 20 to about 60 nucleotides, between about 30 to about 50 nucleotides, or between about 15 to about 30 nucleotides. In some embodiments, the detection oligonucleotide may comprise one or more detectable labels. In some embodiments, the one or more detectable labels comprise a fluorescent moiety. The fluorescent moiety may emit in the red, far-red, near-red, yellow, green, or blue wavelengths. In some embodiments, the fluorescent moiety comprises one or more of 6- FAM (6-carboxyfluorescein), JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein), TAMRA (6-carboxytetramethylrhodamine), 5-Cy5 (5-carboxyrhodamine), 5-Cy5.5 (5- carboxylic acid succinimidyl ester), 5-Cy7 (5-carboxyrhodamine), (hexachlorofluorescein), Alexa Fluor 488 (AF488), Alexa Fluor 514 (AF514), Texas Red, Cyanine 3, Cyanine 5, Pacific Blue, Tetramethyl rhodamine, Oxazole Yellow, Atto647N, and Rhodamine 6G (R6G). In some embodiments, the detection oligonucleotide may comprise two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or 10 or more fluorescent moi eties. In some embodiments, the detection oligonucleotide may comprise 10 or less, nine or less, eight or less, seven or less, six or less, five or less, four or less, three or less, or two or less fluorescent moieties.

[0083]

[0087] In some embodiments, the fluorescent moiety may comprise an organic dye, a biological fluorophore, a quantum dot, or a combination thereof. In some embodiments, the organic dye may comprise an organic molecule. In some embodiments, the organic dye may comprise a coumarin, a cyanine, a benzofuran, a quinoline, a quinazolinone, an indole, a benzazole, a borapolyazaindacene, a xanthene, or a combination thereof. The organic dye may correspond to a color. For example, the organic dye may correspond to a green, a yellow, a blue, an indigo, a red, an orange a purple, a pink, a violet, or a combination thereof. In some embodiments, the organic dye may correspond to no color. In some embodiments, the organic dye may correspond to a black color. In some embodiments, the organic dye may correspond to a white color.

[0084]

[0088] The detectable moiety can be identified by imaging. When the detectable label is a fluorophore, the fluorophore emits a color in the visible light spectrum which can be captured by fluorescent imaging and associated filters. In some embodiments, the fluorophore may emit in a wavelength in the range between 400 nanometers (nm) and 900 nm. In some embodiments, the fluorophore may emit in a wavelength between 400 nm and 475 nm, 475 nm and 490 nm, 490 nm and 530 nm, 530 nm and 575 nm, 575 nm and 600 nm, 600 nm and 700 nm, or 700 nm and 800 nm. In some embodiments, the fluorophore may emit a wavelength of 400 nm or more, 425 nm or more, 450 nm or more, 475 nm or more, 500 nm or more, 525 nm or more, 550 nm or more, 575 nm or more, 600 nm or more, 625 nm or more, 650 nm or more, 675 nm or more, 700 nm or more, 725 nm or more, 750 nm or more, 775 nm or more, 800 nm or more, 825 nm or more, 850 nm or more, 875 nm or more, or 900 nm or more. In some embodiments, the fluorophore may emit a wavelength of 900 nm or less, 875 nm or less, 850 nm or less, 825 nm or less, 800 nm or less, 775 nm or less, 750 nm or less, 725 nm or less, 700 nm or less, 675 nm or less, 650 nm or less, 625 nm or less, 600 nm or less, 575 nm or less, 550 nm or less, 525 nm or less, 500 nm or less, 475 nm or less, 450 nm or less, 425 nm or less, or 400 nm or less.

[0085]

[0089] The detectable labels (e.g., fluorescent moieties) may be optically distinct. The number of optically distinct detectable labels used in the methods described herein can impact the amount of information that is detected. For example, a detection scheme using a larger number of optically distinct detectable labels may allow for a higher amount of multiplexing of codes, which will in turn allow for a greater amount of target molecule related information to be detected and captured. A detection scheme using a smaller number of optically distinct detectable labels would allow for a lesser amount of target molecule related information to be detected and captured. In some embodiments, using a larger number of optically distinct detectable labels may lead to a detection process that identifies a target molecule in less time as compared to using a fewer number of optically distinct detectable labels when querying a concatemeric amplification product. In some embodiments, a detection scheme using a larger number of optically distinct detectable labels may lead to greater instrument complexity, which may lead to fluorescence detection crosstalk, whereby the fluorescence emission spectra of the optically distinct fluorescent moieties may not yield distinct fluorescence signals. In some embodiments, a detection scheme using a greater number of optically distinct fluorescent moieties may require use of a more complex detection tool.

[0086]

[0090] In some embodiments, the detection polynucleotides or detection polynucleotide complexes may be provided in one or more detection pools. In some embodiments, the methods herein may use one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, 45 or more, or 50 or more detection pools. In some embodiments, the methods herein may use 50 or less, 45 or less, 40 or less, 35 or less, 30 or less, 25 or less, 20 or less, 15 or less, ten or less, nine or less, eight or less, seven or less, six or less, five or less, four or less, three or less, or two or less detection pools.

[0091] In some embodiments, each detection pool provided may comprise a number of detection polynucleotides. In some embodiments, each detection pool may comprise two or more, three or more, four or more, five or more, ten or more, 15 or more, 25 or more, 50 or more, 100 or more, 150 or more, 250 or more, 500 or more, 1,000 or more, 1,500 or more, 2,500 or more, or 5,000 or more detection polynucleotides. In some embodiments, each detection pool may comprise 5,000 or less, 2,500 or less, 1,500 or less, 1,000 or less, 500 or less, 250 or less, 150 or less, 100 or less, 50 or less, 25 or less, 15 or less, ten or less, nine or less, eight or less, seven or less, six or less, five or less, four or less, three or less, or two or less detection polynucleotides.

[0087]

[0092] The number of detection pools and the number of detection polynucleotides in each detection pool may be considered in the design of the recognition elements. For example, one advantage to using a smaller number of detection pools and detection polynucleotides in the methods described herein may be to lower design costs. Conversely, one advantage to using a larger number of detection pools and detection polynucleotides in the methods described herein may be the need for a higher degree of multiplexing for target molecule detection and larger amounts of information that may be detected.

[0088]

[0093] The methods described herein include imaging a plurality of detection polynucleotides that have hybridized to their complementary code or a portion thereof in order to obtain identifiable signals which can be correlated back to the presence of a target molecule of interest. In some embodiments, the signals are associated with one or more segments of a code, or portions thereof, for each concatemeric amplification product. In some embodiments, the imaging is performed by an imaging system comprising a fluorescence detection system.

[0089]

[0094] In some embodiments, the imaging is conducted using an imaging system. The imaging system may comprise at the minimum a camera, a detector, an illuminator, a condenser, or a combination thereof. In some embodiments, the imaging may include images of fluorescence emission, luminescence, or a combination thereof. In some embodiments, the imaging systems comprise components or sub-systems of a larger system that may also include optics modules including when needed fluorescence filters, fluidics modules, temperature control modules, translation stages, robotic fluid dispensing and / or microplate handling, processors or computers, instrument control software, data analysis and display software, etc. In some embodiments, the imaging system is a fluorescence imaging system. In some embodiments, the imaging may include fluorescent images from the fluorescent moieties present on the detection polynucleotides.

[0090]

[0095] In some embodiments, the image may comprise fluorescence information from one or more wavelengths. In some embodiments, the fluorescence information may comprise emission data from a wavelength from about 220-830 nanometers (nm), about 230-820 nm, about 240- 810 nm, about 250-800 nm, about 260-790 nm, about 270-780 nm, about 280-770 nm, about 290-760 nm, about 300-750 nm, about 310-740 nm, about 320-730 nm, about 330-720 nm, about 340-710 nm, about 350-700 nm, about 360-690 nm, about 370-680 nm, about 380-670 nm, about 390-660 nm, about 400-650 nm, about 410-640 nm, about 420-630 nm, about 430- 620 nm, about 440-610 nm, about 450-600 nm, about 460-590 nm, about 470-580nm, about 480-570 nm, about 490-560 nm, about 500-550 nm, about 510-540 nm, about 520-530 nm, or a combination thereof.

[0091]

[0096] Image detection and capture may relate to iteratively repeating the operations of: (i) introducing detection polynucleotides to the concatemeric amplification products; (ii) hybridizing the detection polynucleotides to a code or a portion thereof; and (iii) imaging the signals of the hybridized detection polynucleotides to a code or a portion thereof. In some embodiments, the iterative repetition of the operations is performed for each nucleic acid segment of a code, or a portion thereof, one or more times.

[0092]

[0097] In some embodiments, the iteratively repeating the operations of: (i) introducing detection polynucleotides to the concatemeric amplification products; (ii) hybridizing the detection polynucleotides to a code or a portion thereof; and (iii) imaging the signals of the hybridized detection polynucleotides to a code or a portion thereof comprises two or more iterative repetitions. For example, the methods described herein may comprise 2-50 iterative repetitions, 2-10 iterative repetitions, or 2-8 iterative repetitions. In some embodiments, the method described herein may comprise one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or 50 or more iterative repetitions of : (i) introducing detection polynucleotides to the concatemeric amplification products; (ii) hybridizing the detection polynucleotides to a code or a portion thereof; and (iii) imaging the signals of the hybridized detection polynucleotides to a code or a portion thereof.

[0098] In some embodiments, the number of iterative repetitions of the operations of(i) introducing detection polynucleotides to the concatemeric amplification products; (ii) hybridizing the detection polynucleotides to a code or a portion thereof; and (iii) imaging the signals of the hybridized detection polynucleotides to a code or a portion thereof corresponds to the number of nucleic acid segments present in a code of the recognition element. In some embodiments, each nucleic acid segment of the code of the recognition element may undergo a number of iterative repetitions of the operations of: (i) introducing detection polynucleotides to the concatemeric amplification products; (ii) hybridizing the detection polynucleotides to a code or a portion thereof; and (iii) imaging the signals of the hybridized detection polynucleotides to a code or a portion thereof. For example, the methods described herein may comprise iteratively repeating the operations two times per segment, three times per segment, or four times per segment. In some embodiments, the methods described herein may comprise iteratively repeating the operations two or more times per segment, three or more times per segment, four or more times per segment, five or more times per segment, six or more times per segment, seven or more times per segment, eight or more times per segment, nine or more times per segment, 10 or more times per segment, 11 or more times per segment, 12 or more times per segment, 13 or more times per segment, 14 or more times per segment, 15 or more times per segment, 16 or more times per segment, 17 or more times per segment, 18 or more times per segment, 19 or more times per segment, or 20 or more times per segment.

[0093]

[0099] The iterative process results in a fluorescent profile, a code profile, which can be analyzed, decoded and matched to a code in a recognition element thereby identifying the presence of the target of interest.

[0094]

[0100] Additional methods for detection comprising detection polynucleotides, imaging detection polynucleotides, and decoding the code profile by soft decision decoding can be found in WO2023 / 158993 A2 and PCT / US2024 / 62056, which is incorporated herein by reference in their entireties.

[0095]

[0101] Several models may be used to finally identify a code that is associated with a target molecule based on the fluorescence signals generated, images captured, and a code profile generated from detection polynucleotide hybridization to code sequences. In one embodiment, decoding makes use of a hard decision decoding model. In another embodiment, decoding makes use of a soft decision decoding model.

[0096]

[0102] For soft decision decoding, it is not necessary to identify each base specifically. For example, signals generated during each detection event may be detected and recorded to produce a data set that may be used as input into a model to calculate a probability that a specific code is present without requiring that each base of a code be determined. Although it is not necessary in a soft decision decoding model to make a hard decision about the identity of each nucleotide, a model could nevertheless include assigning a probability or identity to each nucleotide in the sequence of a code, wherein each nucleotide in the sequence of a code could be sequenced. Data gathered includes intensity readings for signals produced by the hybridized detection polynucleotide fluorescent moiety in various spectral bands. A set of intensity readings are detected by imaging, stored and used as input into a soft decision decoding model for determining a probability that a particular code is present, and hence a target nucleic acid is present in the sample.

[0097]

[0103] A model may be developed or trained using data from known codes, such as signal intensity data across a predetermined spectrum. The model may be used to calculate a set of probabilities across a set of one or more codes, indicating, for example, for each code, a probability that it is present in a concatemeric amplification product.

[0098]

[0104] The probability that a particular code is present is indicative of the probability that a particular target molecule associated with the code is present in the sample of interest. Data indicating the probability that a particular target is present is, for example, to calculate probabilities relevant to diagnosis or screening of various medical conditions, or selection of drugs for treatment of various medical conditions.

[0099]

[0105] A soft decoding decision model comprises using an algorithm to predict the presence of target molecules from a sample. In some embodiments, the algorithm is a soft-decision decoding algorithm. In some embodiments, the algorithm is applied to the codes of the concatemeric amplification products for predicting the presence of a target molecule from a sample.

[0100]

[0106] The methods disclosed herein may comprise soft decision decoding to predict the presence of the code in a recognition element or concatemeric amplification product thereof, wherein the presence of the code correlates and serves as a proxy for the presence of a target nucleic acid in a sample. In some embodiments, the methods described herein may use soft decision decoding. In some embodiments, the methods described herein may use hard decision decoding. For hard decision decoding, signals from queried concatemers are extracted from images. This is the same for soft decision decoding, in that signals that are generated and imaged are extracted from the images. For hard decision decoding, hard basecalls are generated from the intensities of the signals, whereas with soft decision decoding no hard basecalls are necessary as all of the signal range is retained. The code assignment for hard decision decoding is determined by matching nucleotide reads to codes, whereas with soft decision decoding, the signals are cross correlated against the expects signals and the most likely code is assigned, as such a probabilistic methodology. When using soft decision decoding, it is not necessary for the model to identify each base specifically. For example, signals (e.g., fluorescent signals) generated during each cycle of a detection process may be detected and recorded to produce a data set that may be used as input into a model to calculate a probability that a specific code is present.

[0101]

[0107] The permutation space on a recognition element is the totality of factors that determines the number of unique nucleotide possibilities at each nucleic acid segment. Factors comprise the number of segments present on a recognition element, the number of incubation periods or times a segment is queried with a detection pool of detection polynucleotides, and the number of computational symbols or colors.

[0102]

[0108] Fig. 4 details an example of a soft decision decoding pipeline for determining the presence of a target molecule from a sample based on detection and decoding of a code associated with the target molecule that originally hybridized to a recognition element. Referring to the example workflow in Fig. 4, images of the sample are acquired, aligned, and processed to extract the intensity of the features, or signals of interest across the imaged field of view in multiple spectral channels, thereby generating a code profile. The corrected intensities of said features are fed through a series of algorithms that make up the soft decoder. At first, the intensity profiles of the codes are learned based on features of high confidence or high intensity. This trained model provides a template for each code from which the rest of the features of interest are compared to in the second operation. Third, a confidence score is computed from the difference between the intensity profile of each feature and the trained profiles. Several filters are then applied to remove outliers, duplicates, and low confidence decoded concatemers. The final output is a table of decoded concatemers with associated filter status, confidence score, and most likely assignment to one of the codes of one or more concatemeric amplification products.

[0109] In some embodiments, a recognition element comprises a larger code, for example a code with four segments instead of two or three. In some embodiments, a recognition element comprising a larger code may result in a detection scheme with better error correction. Additionally, in some embodiments, a larger code may result in a lower signal -to-noise ratio. [HO] In some embodiments, a recognition element comprises a smaller code, for example a code with two segments, or one segment. In some embodiments, a recognition element comprising a small code may result in a detection scheme with lower error correction abilities. Further, in some embodiments, a small code may result in a higher signal-to-noise ratio.

[0103] [Hl] Disclosed herein, in some embodiments, are systems comprising one or more assay components, one or more detection systems or devices, one or more kit components, a computer system, or a combination thereof. In some embodiments, the systems comprise a solid substrate configured to immobilize a circularized recognition element, a concatemeric amplification product, a detection polynucleotide, or a hybridized complex of a concatemeric amplification product and a detection polynucleotide complex. In some embodiments, the systems comprise a welled plate or a flowcell. In some embodiments, the systems comprise a fluid flow controller, a temperature controller, an imaging system, a computer system, or any combination thereof.

[0112] In some embodiments, the systems disclosed herein may include a solid substrate or a solid surface. The solid substrates and surfaces disclosed herein may be referred to as a substrate, a support, a solid support, or a surface. The substrate may be modified for immobilizing circularized and ligated recognition elements or concatemeric amplification products, or both. Examples of solid substrates include, but are not limited to, glass, modified or functionalized glass, plastics, polysaccharides, nylon, nitrocellulose, ceramics, resins, silica, silica-based materials, carbon, metals, inorganic glasses, plastics, optical fiber bundles, optically clear glass, and other polymers. In some embodiments, the plastic solid substrates may include acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, or polyurethanes. In some embodiments, the silica-based solid substrates may include silicon or modified silicon.

[0104]

[0113] In some embodiments, the substrate may be a welled plate. In some embodiments, the substrate may be a 96-well plate. In some embodiments, the substrate may be a 4-well plate, a 6- well plate, an 8-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 384-well plate, an 864-well plate, or a 1,536-well plate. In some embodiments, the substrate may have greater than or equal to 96 wells. In some embodiments, the substrate may have less than or equal to 96 wells.

[0105]

[0114] In some embodiments, the substrate may be a flowcell. In some embodiments, the flowcell may have two or more lanes. In some embodiments, the flowcell may have two or less lanes.

[0106]

[0115] In some embodiments, the substrate may be a microarray, a slide, a chip, a microwell, a tube, a column, a particle, a bead, or a paramagnetic bead.

[0107]

[0116] In some embodiments, the substrate may comprise a coating. In some embodiments, the coating may comprise a layer that may be charged. In some embodiments, the coating layer may be positively charged. In some embodiments, the coating layer may be negatively charged. In some embodiments, the coating may be non-charged. In some embodiments, the substrate may comprise a surface comprising a cation-coating layer. In some embodiments, the substrate may comprise a surface comprising an anion-coating layer. In some embodiments, the substrate may comprise a surface comprising a neutral-charged layer. In some embodiments, the substrate may be coated with streptavidin. In some embodiments, the substrate may be coated with avidin. In some embodiments, the substrate may be coated with one or more antibodies.

[0108]

[0117] The systems disclosed herein may comprise a fluidics system. The fluidics system may comprise a fluid flow controller. In some embodiments, the fluid flow controller may comprise one or more pumps, valves, mixing manifolds, reagent reservoirs, waste reservoirs, or any combination thereof. In some embodiments, the fluidic system and subcomponents of the fluidics system are fluidically connected to the reaction vessel of the present disclosure. In some embodiments, the fluidic system and subcomponents of the fluidics system iteratively flow in reagents (e.g., buffers, detector polynucleotides, anchor polynucleotides, detection oligonucleotide complexes, etc.) to the reaction vessel. In some embodiments, the reaction vessel comprises a solid substrate configured to immobilize the circularized and ligated recognition elements or concatemeric amplification products thereof.

[0109]

[0118] The systems disclosed herein may comprise a temperature system. The temperature system may comprise a temperature controller. The temperature controller may be incorporated into the systems described herein to facilitate accuracy of the methods and systems described herein. In some embodiments, the temperature controller may comprise temperature control components. Non-limiting examples of temperature control components include resistive heating elements, infrared light sources, heating or cooling devices, heat sinks, thermocouples, thermistors, or a combination thereof. In some embodiments, the temperature controller may provide changes in temperature over specified time intervals. In some embodiments, the temperature controller may provide an increase in temperature. In some embodiments, the temperature controller may provide a decrease in temperature. In some embodiments, the temperature controller may provide for cycling of temperatures between two or more set temperatures so that thermocycling or amplification may be performed. In some embodiments, the temperature controller may provide a constant temperature.

[0110]

[0119] The systems disclosed herein may comprise an imaging system. In some embodiments, signals produced by the detection polynucleotides disclosed herein may be imaged by the imaging systems disclosed herein. The imaging system may comprise one or more light sources, one or more optical components, one or more filters, one or more imaging sensors for imaging and detection, or a combination thereof. In some embodiments, the one or more light sources may comprise light from a bulb. In some embodiments, the one or more optical components may comprise lenses, mirrors, digital mirror devices, prisms, optical filters, colored glass filters, narrowband interference filters, broadband interference filters, dichroic reflectors, diffraction gratings, apertures, optical fibers, optical waveguides, or a combination thereof. In some embodiments, the one or more imaging sensors may comprise a charge-coupled device (CCD) sensor or camera, a complementary metal -oxide-semiconductor (CMOS) imaging sensor or camera, a negative-channel metal-oxide semiconductor (NMOS) imaging sensor or camera, or a combination thereof.

[0111]

[0120] Various operations of the methods and systems disclosed herein may be performed by a computer system of the present disclosure. Referring to Fig. 5, a block diagram is shown depicting an example machine that includes a computer system 500 (e.g., a processing or computing system) within which a set of instructions can execute for causing a device to perform or execute any one or more of the aspects and / or methodologies for static code scheduling of the present disclosure. The components in Fig. 5 are examples only and do not limit the scope of use or functionality of any hardware, software, embedded logic component, or a combination of two or more such components implementing particular embodiments.

[0112]

[0121] Computer system 500 may include one or more processors 501, a memory 503, and a storage 508 that communicate with each other, and with other components, via a bus 540 (not shown). The bus 540 may also link a display 532, one or more input devices 533 (which may, for example, include a keypad, a keyboard, a mouse, a stylus, etc.), one or more output devices

[0113] 534, one or more storage devices 535, and various tangible storage media 536. All of these elements may interface directly or via one or more interfaces or adaptors to the bus 540. For instance, the various tangible storage media 536 can interface with the bus 540 via storage medium interface 526. Computer system 500 may have any suitable physical form, including but not limited to one or more integrated circuits (ICs), printed circuit boards (PCBs), mobile handheld devices (such as mobile telephones or PDAs), laptop or notebook computers, distributed computer systems, computing grids, or servers.

[0114]

[0122] Computer system 500 may include one or more processor(s) 501 (e.g., central processing units (CPUs), general purpose graphics processing units (GPGPUs), or quantum processing units (QPUs)) that carry out functions. Processor(s) 501 optionally may contain a cache memory unit

[0115] 502 for temporary local storage of instructions, data, or computer addresses. Processor(s) 501 are configured to assist in execution of computer readable instructions. Computer system 500 may provide functionality for the components depicted in Fig. 5 as a result of the processor(s) 501 executing non-transitory, processor-executable instructions embodied in one or more tangible computer-readable storage media, such as memory 503, storage 508, storage devices

[0116] 535, and / or storage medium 536. The computer-readable media may store software that implements particular embodiments, and processor(s) 501 may execute the software. Memory

[0117] 503 may read the software from one or more other computer-readable media (such as mass storage device(s) 535, 536) or from one or more other sources through a suitable interface, such as network interface 520. The software may cause processor(s) 501 to carry out one or more processes or one or more operations of one or more processes described or illustrated herein. Carrying out such processes or operations may include defining data structures stored in memory 503 and modifying the data structures as directed by the software.

[0118]

[0123] The memory 503 may include various components (e.g., machine readable media) including, but not limited to, a random-access memory component (e.g., RAM 504) (e.g., static RAM (SRAM), dynamic RAM (DRAM), ferroelectric random access memory (FRAM), phasechange random access memory (PRAM), etc.), a read-only memory component (e.g., ROM 505), and any combinations thereof. ROM 505 may act to communicate data and instructions unidirectionally to processor(s) 501, and RAM 504 may act to communicate data and instructions bidirectionally with processor(s) 501. ROM 505 and RAM 504 may include any suitable tangible computer-readable media described below. In one example, a basic input / output system 506 (BIOS), including basic routines that help to transfer information between elements within computer system 500, such as during start-up, may be stored in the memory 503.

[0124] Fixed storage 508 is connected bidirectionally to processor(s) 501, optionally through storage control unit 507. Fixed storage 508 provides additional data storage capacity and may also include any suitable tangible computer-readable media described herein. Storage 508 may be used to store operating system 509, executable(s) 510, data 511, applications 512 (application programs), and the like. Storage 508 can also include an optical disk drive, a solid-state memory device (e.g., flash-based systems), or a combination of any of the above. Information in storage 508 may, in appropriate cases, be incorporated as virtual memory in memory 503.

[0119]

[0125] In one example, storage device(s) 535 may be removably interfaced with computer system 500 (e.g., via an external port connector (not shown)) via a storage device interface 525. Particularly, storage device(s) 535 and an associated machine-readable medium may provide non-volatile and / or volatile storage of machine-readable instructions, data structures, program modules, and / or other data for the computer system 500. In one example, software may reside, completely or partially, within a machine-readable medium on storage device(s) 535. In another example, software may reside, completely or partially, within processor(s) 501.

[0120]

[0126] Bus 540 connects a wide variety of subsystems. Herein, reference to a bus may encompass one or more digital signal lines serving a common function, where appropriate. Bus 540 may be any of several types of bus structures including, but not limited to, a memory bus, a memory controller, a peripheral bus, a local bus, and any combinations thereof, using any of a variety of bus architectures. As an example, and not by way of limitation, such architectures include an Industry Standard Architecture (ISA) bus, an Enhanced ISA (EISA) bus, a Micro Channel Architecture (MCA) bus, a Video Electronics Standards Association local bus (VLB), a Peripheral Component Interconnect (PCI) bus, a PCI-Express (PCI-X) bus, an Accelerated Graphics Port (AGP) bus, HyperTransport (HTX) bus, serial advanced technology attachment (SATA) bus, and any combinations thereof.

[0121]

[0127] Computer system 500 may also include an input device 533. In one example, a user of computer system 500 may enter commands and / or other information into computer system 500 via input device(s) 533. Examples of an input device(s) 533 include, but are not limited to, an alpha-numeric input device (e.g., a keyboard), a pointing device (e.g., a mouse or touchpad), a touchpad, a touch screen, a multi-touch screen, a joystick, a stylus, a gamepad, an audio input device (e.g., a microphone, a voice response system, etc.), an optical scanner, a video or still image capture device (e.g., a camera), and any combinations thereof. In some embodiments, the input device is a Kinect, Leap Motion, or the like. Input device(s) 533 may be interfaced to bus 540 via any of a variety of input interfaces 523 (e.g., input interface 523) including, but not limited to, serial, parallel, game port, USB, FIREWIRE, THUNDERBOLT, or any combination of the above.

[0128] In particular embodiments, when computer system 500 is connected to network 530, computer system 500 may communicate with other devices, specifically mobile devices and enterprise systems, distributed computing systems, cloud storage systems, cloud computing systems, and the like, connected to network 530. Communications to and from computer system 500 may be sent through network interface 520. For example, network interface 520 may receive incoming communications (such as requests or responses from other devices) in the form of one or more packets (such as Internet Protocol (IP) packets) from network 530, and computer system 500 may store the incoming communications in memory 503 for processing. Computer system 500 may similarly store outgoing communications (such as requests or responses to other devices) in the form of one or more packets in memory 503 and communicated to network 530 from network interface 520. Processor(s) 501 may access these communication packets stored in memory 503 for processing.

[0122]

[0129] Examples of the network interface 520 include, but are not limited to, a network interface card, a modem, and any combination thereof. Examples of a network 530 or network segment 530 include, but are not limited to, a distributed computing system, a cloud computing system, a wide area network (WAN) (e.g., the Internet, an enterprise network), a local area network (LAN) (e.g., a network associated with an office, a building, a campus or other relatively small geographic space), a telephone network, a direct connection between two computing devices, a peer-to-peer network, and any combinations thereof. A network, such as network 530, may employ a wired and / or a wireless mode of communication. In general, any network topology may be used.

[0123]

[0130] Information and data can be displayed through a display 532. Examples of a display 532 include, but are not limited to, a cathode ray tube (CRT), a liquid crystal display (LCD), a thin film transistor liquid crystal display (TFT-LCD), an organic liquid crystal display (OLED) such as a passive-matrix OLED (PMOLED) or active-matrix OLED (AMOLED) display, a plasma display, and any combinations thereof. The display 532 can interface to the processor(s) 501, memory 503, and fixed storage 508, as well as other devices, such as input device(s) 533, via the bus 540. The display 532 is linked to the bus 540 via a video interface 522, and transport of data between the display 532 and the bus 540 can be controlled via the graphics control 521. In some embodiments, the display is a video projector. In some embodiments, the display is a headmounted display (HMD) such as a VR headset. In further embodiments, suitable VR headsets include, by way of non-limiting examples, HTC Vive, Oculus Rift, Samsung Gear VR, Microsoft HoloLens, Razer OSVR, FOVE VR, Zeiss VR One, Avegant Glyph, Freefly VR headset, and the like. In still further embodiments, the display is a combination of devices such as those disclosed herein.

[0131] In addition to a display 532, computer system 500 may include one or more other peripheral output devices 534 including, but not limited to, an audio speaker, a printer, a storage device, and any combinations thereof. Such peripheral output devices may be connected to the bus 540 via an output interface 524. Examples of an output interface 524 include, but are not limited to, a serial port, a parallel connection, a USB port, a FIREWIRE port, a THUNDERBOLT port, and any combinations thereof.

[0124]

[0132] In addition, or as an alternative, computer system 500 may provide functionality as a result of logic hardwired or otherwise embodied in a circuit, which may operate in place of or together with software to execute one or more processes or one or more operations of one or more processes described or illustrated herein. Reference to software in this disclosure may encompass logic, and reference to logic may encompass software. Moreover, reference to a computer-readable medium may encompass a circuit (such as an IC) storing software for execution, a circuit embodying logic for execution, or both, where appropriate. The present disclosure encompasses any suitable combination of hardware, software, or both.

[0125]

[0133] Those of skill in the art will appreciate that the various illustrative logical blocks, modules, circuits, and algorithm operations described in connection with the embodiments disclosed herein may be implemented as electronic hardware, computer software, or combinations of both. To clearly illustrate this interchangeability of hardware and software, various illustrative components, blocks, modules, circuits, and operations have been described above generally in terms of their functionality.

[0126]

[0134] The various illustrative logical blocks, modules, and circuits described in connection with the embodiments disclosed herein may be implemented or performed with a general- purpose processor, a digital signal processor (DSP), an application specific integrated circuit (ASIC), a field programmable gate array (FPGA) or other programmable logic device, discrete gate or transistor logic, discrete hardware components, or any combination thereof designed to perform the functions described herein. A general -purpose processor may be a microprocessor, but in the alternative, the processor may be any conventional processor, controller, microcontroller, or state machine. A processor may also be implemented as a combination of computing devices, e.g., a combination of a DSP and a microprocessor, a plurality of microprocessors, one or more microprocessors in conjunction with a DSP core, or any other such configuration.

[0127]

[0135] The operations of a method or algorithm described in connection with the embodiments disclosed herein may be embodied directly in hardware, in a software module executed by one or more processor(s), or in a combination of the two. A software module may reside in RAM memory, flash memory, ROM memory, EPROM memory, EEPROM memory, registers, hard disk, a removable disk, a CD-ROM, or any other form of storage medium known in the art. An exemplary storage medium is coupled to the processor such the processor can read information from, and write information to, the storage medium. In the alternative, the storage medium may be integral to the processor. The processor and the storage medium may reside in an ASIC. The ASIC may reside in a user terminal. In the alternative, the processor and the storage medium may reside as discrete components in a user terminal.

[0128]

[0136] In accordance with the description herein, suitable computing devices include, by way of non-limiting examples, server computers, desktop computers, laptop computers, notebook computers, sub-notebook computers, netbook computers, notepad computers, set-top computers, media streaming devices, handheld computers, Internet appliances, mobile smartphones, tablet computers, personal digital assistants, video game consoles, and vehicles. Those of skill in the art will also recognize that select televisions, video players, and digital music players with optional computer network connectivity are suitable for use in the system described herein. Suitable tablet computers, in various embodiments, include those with booklet, slate, and convertible configurations, known to those of skill in the art.

[0129]

[0137] In some embodiments, the computing device includes an operating system configured to perform executable instructions. The operating system is, for example, software, including programs and data, which manages the device’s hardware and provides services for execution of applications. Those of skill in the art will recognize that suitable server operating systems include, by way of non-limiting examples, FreeBSD, OpenBSD, NetBSD®, Linux, Apple® Mac OS X Server®, Oracle® Solaris®, Windows Server®, and Novell® NetWare®. Those of skill in the art will recognize that suitable personal computer operating systems include, by way of nonlimiting examples, Microsoft® Windows®, Apple® Mac OS X®, UNIX®, and UNIX-like operating systems such as GNU / Linux®. In some embodiments, the operating system is provided by cloud computing. Those of skill in the art will also recognize that suitable mobile smartphone operating systems include, by way of non-limiting examples, Nokia® Symbian® OS, Apple® iOS®, Research In Motion® BlackBerry OS®, Google® Android®, Microsoft® Windows Phone® OS, Microsoft® Windows Mobile® OS, Linux®, and Palm® WebOS®. Those of skill in the art will also recognize that suitable media streaming device operating systems include, by way of non-limiting examples, Apple TV®, Roku®, Boxee®, Google TV®, Google Chromecast®, Amazon Fire®, and Samsung® HomeSync®. Those of skill in the art will also recognize that suitable video game console operating systems include, by way of non-limiting examples, Sony® PS3®, Sony® PS4®, Microsoft® Xbox 360®, Microsoft Xbox One, Nintendo® Wii®, Nintendo® Wii U®, and Ouya®.

[0138] In some embodiments, the platforms, systems, media, and methods disclosed herein include one or more non-transitory computer readable storage media encoded with a program including instructions executable by the operating system of an optionally networked computing device. In further embodiments, a computer readable storage medium is a tangible component of a computing device. In further embodiments, a computer readable storage medium is optionally removable from a computing device. In some embodiments, a computer readable storage medium includes, by way of non-limiting examples, CD-ROMs, DVDs, flash memory devices, solid state memory, magnetic disk drives, magnetic tape drives, optical disk drives, distributed computing systems including cloud computing systems and services, and the like. In some cases, the program and instructions are permanently, substantially permanently, semi-permanently, or non-transitorily encoded on the media.

[0130]

[0139] In some embodiments, the platforms, systems, media, and methods disclosed herein include at least one computer program, or use of the same. A computer program includes a sequence of instructions, executable by one or more processor(s) of the computing device’s CPU, written to perform a specified task. Computer readable instructions may be implemented as program modules, such as functions, objects, Application Programming Interfaces (APIs), computing data structures, and the like, which perform particular tasks or implement particular abstract data types. In light of the disclosure provided herein, those of skill in the art will recognize that a computer program may be written in various versions of various languages.

[0131]

[0140] The functionality of the computer readable instructions may be combined or distributed as desired in various environments. In some embodiments, a computer program comprises one sequence of instructions. In some embodiments, a computer program comprises a plurality of sequences of instructions. In some embodiments, a computer program is provided from one location. In other embodiments, a computer program is provided from a plurality of locations. In various embodiments, a computer program includes one or more software modules. In various embodiments, a computer program includes, in part or in whole, one or more web applications, one or more mobile applications, one or more standalone applications, one or more web browser plug-ins, extensions, add-ins, or add-ons, or combinations thereof.

[0132]

[0141] In some embodiments, the computer programs described herein may be used to perform at least one function. The computer programs described herein may perform functions related to storing data, receiving data, analyzing data, exporting data, or a combination thereof. In some embodiments, the computer programs described herein may perform functions related to applying selection criteria, including in silico selection criteria, functional selection criteria, or a combination thereof. In some embodiments, the computer programs may receive sequence information, including sequence information for nucleic acid segments. The sequence information may be configured as an array, a table, a list, or combination thereof. The sequence information may be formatted in a variety of ways, including, but not limited to a .txt file, a FASTA file, an .xls file, or a combination thereof. The computer programs described herein may apply selection criterion or selection criteria to a set of nucleic acid segments. The computer programs may sort the nucleic acid segments, determine or compute characteristics of the nucleic acid segments, perform calculations, reorder the nucleic acid segments, or a combination thereof. In some embodiments, the computer programs described herein may store information related to the nucleic acid segments. In some embodiments, the computer program may use information stored related to the nucleic acid segments to apply selection criteria to the nucleic acid segments. In certain embodiments, the computer program may receive information and / or data related to nucleic acid segments, selection criteria, or a combination thereof. In some embodiments, the computer programs may perform functions related to analyzing data from functional assays, including, but not limited to functional assays described herein. In some embodiments, analyzing data from functional assays may comprise image analysis, image quantification, intensity quantification, feature identification, or a combination thereof. The computer programs described herein may also export information. In some embodiments, the exported information may comprise images, files, data tables, documents, folders, or a combination thereof.

[0133]

[0142] In some embodiments, a computer program includes a web application. In light of the disclosure provided herein, those of skill in the art will recognize that a web application, in various embodiments, utilizes one or more software frameworks and one or more database systems. In some embodiments, a web application is created upon a software framework such as Microsoft® .NET or Ruby on Rails (RoR). In some embodiments, a web application utilizes one or more database systems including, by way of non-limiting examples, relational, non-relational, object oriented, associative, XML, and document oriented database systems. In further embodiments, suitable relational database systems include, by way of non-limiting examples, Microsoft® SQL Server, mySQL™, and Oracle®. Those of skill in the art will also recognize that a web application, in various embodiments, is written in one or more versions of one or more languages. A web application may be written in one or more markup languages, presentation definition languages, client-side scripting languages, server-side coding languages, database query languages, or combinations thereof. In some embodiments, a web application is written to some extent in a markup language such as Hypertext Markup Language (HTML), Extensible Hypertext Markup Language (XHTML), or extensible Markup Language (XML). In some embodiments, a web application is written to some extent in a presentation definition language such as Cascading Style Sheets (CSS). In some embodiments, a web application is written to some extent in a client-side scripting language such as Asynchronous JavaScript and XML (AJAX), Flash® ActionScript, JavaScript, or Silverlight®. In some embodiments, a web application is written to some extent in a server-side coding language such as Active Server Pages (ASP), ColdFusion®, Perl, Java™, JavaServer Pages (JSP), Hypertext Preprocessor (PHP), Python™, Ruby, Tel, Smalltalk, WebDNA®, or Groovy. In some embodiments, a web application is written to some extent in a database query language such as Structured Query Language (SQL). In some embodiments, a web application integrates enterprise server products such as IBM® Lotus Domino®. In some embodiments, a web application includes a media player element. In various further embodiments, a media player element utilizes one or more of many suitable multimedia technologies including, by way of non-limiting examples, Adobe® Flash®, HTML 5, Apple® QuickTime®, Microsoft® Silverlight®, Java™, and Unity®.

[0134]

[0143] Referring to Fig. 6, in a particular embodiment, an application provision system comprises one or more databases 600 accessed by a relational database management system (RDBMS) 610. Suitable RDBMSs include Firebird, MySQL, PostgreSQL, SQLite, Oracle Database, Microsoft SQL Server, IBM DB2, IBM Informix, SAP Sybase, Teradata, and the like. In this embodiment, the application provision system further comprises one or more application severs 620 (such as Java servers, .NET servers, PHP servers, and the like) and one or more web servers 630 (such as Apache, IIS, GWS and the like). The web server(s) optionally expose one or more web services via app application programming interfaces (APIs) 640. Via a network, such as the Internet, the system provides browser-based and / or mobile native user interfaces.

[0135]

[0144] Referring to Fig. 7, in a particular embodiment, an application provision system alternatively has a distributed, cloud-based architecture 700 and comprises elastically load balanced, auto-scaling web server resources 710 and application server resources 720 as well as synchronously replicated databases 730.

[0136]

[0145] In some embodiments, a computer program includes a mobile application provided to a mobile computing device. In some embodiments, the mobile application is provided to a mobile computing device at the time it is manufactured. In other embodiments, the mobile application is provided to a mobile computing device via the computer network described herein.

[0137]

[0146] In view of the disclosure provided herein, a mobile application is created by techniques known to those of skill in the art using hardware, languages, and development environments known to the art. Those of skill in the art will recognize that mobile applications are written in several languages. Suitable programming languages include, by way of non-limiting examples, C, C++, C#, Objective-C, Java™, JavaScript, Pascal, Object Pascal, Python™, Ruby, VB.NET, WML, and XHTML / HTML with or without CSS, or combinations thereof.

[0147] Suitable mobile application development environments are available from several sources. Commercially available development environments include, by way of non-limiting examples, AirplaySDK, alcheMo, Appcelerator®, Celsius, Bedrock, Flash Lite, .NET Compact Framework, Rhomobile, and WorkLight Mobile Platform. Other development environments are available without cost including, by way of non-limiting examples, Lazarus, MobiFlex, MoSync, and Phonegap. Also, mobile device manufacturers distribute software developer kits including, by way of non-limiting examples, iPhone and iPad (iOS) SDK, Android™ SDK, BlackBerry® SDK, BREW SDK, Palm® OS SDK, Symbian SDK, webOS SDK, and Windows® Mobile SDK.

[0138]

[0148] Those of skill in the art will recognize that several commercial forums are available for distribution of mobile applications including, by way of non-limiting examples, Apple® App Store, Google® Play, Chrome WebStore, BlackBerry® App World, App Store for Palm devices, App Catalog for webOS, Windows® Marketplace for Mobile, Ovi Store for Nokia® devices, Samsung® Apps, and Nintendo® DSi Shop.

[0139]

[0149] In some embodiments, a computer program includes a standalone application, which is a program that is run as an independent computer process, not an add-on to an existing process, e.g., not a plug-in. Those of skill in the art will recognize that standalone applications are often compiled. A compiler is a computer program(s) that transforms source code written in a programming language into binary object code such as assembly language or machine code. Suitable compiled programming languages include, by way of non-limiting examples, C, C++, Objective-C, COBOL, Delphi, Eiffel, Java™, Lisp, Python™, Visual Basic, and VB .NET, or combinations thereof. Compilation is often performed, at least in part, to create an executable program. In some embodiments, a computer program includes one or more executable complied applications.

[0140]

[0150] In some embodiments, the computer program includes a web browser plug-in (e.g., extension, etc.). In computing, a plug-in is one or more software components that add specific functionality to a larger software application. Makers of software applications support plug-ins to enable third-party developers to create abilities which extend an application, to support easily adding new features, and to reduce the size of an application. When supported, plug-ins enable customizing the functionality of a software application. For example, plug-ins are commonly used in web browsers to play video, generate interactivity, scan for viruses, and display particular file types. Those of skill in the art will be familiar with several web browser plug-ins including, Adobe® Flash® Player, Microsoft® Silverlight®, and Apple® QuickTime®. In some embodiments, the toolbar comprises one or more web browser extensions, add-ins, or add-ons. In some embodiments, the toolbar comprises one or more explorer bars, tool bands, or desk bands.

[0141]

[0151] In view of the disclosure provided herein, those of skill in the art will recognize that several plug-in frameworks are available that enable development of plug-ins in various programming languages, including, by way of non-limiting examples, C++, Delphi, Java™, PHP, Python™, and VB .NET, or combinations thereof.

[0142]

[0152] Web browsers (also called Internet browsers) are software applications, designed for use with network-connected computing devices, for retrieving, presenting, and traversing information resources on the World Wide Web. Suitable web browsers include, by way of nonlimiting examples, Microsoft® Internet Explorer®, Mozilla® Firefox®, Google® Chrome, Apple® Safari®, Opera Software® Opera®, and KDE Konqueror. In some embodiments, the web browser is a mobile web browser. Mobile web browsers (also called microbrowsers, mini -browsers, and wireless browsers) are designed for use on mobile computing devices including, by way of nonlimiting examples, handheld computers, tablet computers, netbook computers, subnotebook computers, smartphones, music players, personal digital assistants (PDAs), and handheld video game systems. Suitable mobile web browsers include, by way of non-limiting examples, Google® Android® browser, RIM BlackBerry® Browser, Apple® Safari®, Palm® Blazer, Palm® WebOS® Browser, Mozilla® Firefox® for mobile, Microsoft® Internet Explorer® Mobile, Amazon® Kindle® Basic Web, Nokia® Browser, Opera Software® Opera® Mobile, and Sony® PSP™ browser.

[0143]

[0153] In some embodiments, the platforms, systems, media, and methods disclosed herein include software, server, and / or database modules, or use of the same. In view of the disclosure provided herein, software modules are created by techniques known to those of skill in the art using machines, software, and languages known to the art. The software modules disclosed herein are implemented in a multitude of ways. In various embodiments, a software module comprises a file, a section of code, a programming object, a programming structure, a distributed computing resource, a cloud computing resource, or combinations thereof. In further various embodiments, a software module comprises a plurality of files, a plurality of sections of code, a plurality of programming objects, a plurality of programming structures, a plurality of distributed computing resources, a plurality of cloud computing resources, or combinations thereof. In various embodiments, the one or more software modules comprise, by way of nonlimiting examples, a web application, a mobile application, a standalone application, and a distributed or cloud computing application. In some embodiments, software modules are in one computer program or application. In other embodiments, software modules are in more than one computer program or application. In some embodiments, software modules are hosted on one machine. In other embodiments, software modules are hosted on more than one machine. In further embodiments, software modules are hosted on a distributed computing platform such as a cloud computing platform. In some embodiments, software modules are hosted on one or more machines in one location. In other embodiments, software modules are hosted on one or more machines in more than one location.

[0144]

[0154] In some embodiments, the platforms, systems, media, and methods disclosed herein include one or more databases, or use of the same. In view of the disclosure provided herein, those of skill in the art will recognize that many databases are suitable for storage and retrieval of nucleic acid segment sequences or analysis thereof information. In various embodiments, suitable databases include, by way of non-limiting examples, relational databases, non-relational databases, object oriented databases, object databases, entity-relationship model databases, associative databases, XML databases, document oriented databases, and graph databases. Further non-limiting examples include SQL, PostgreSQL, MySQL, Oracle, DB2, Sybase, and MongoDB. In some embodiments, a database is Internet-based. In further embodiments, a database is web-based. In still further embodiments, a database is cloud computing-based. In a particular embodiment, a database is a distributed database. In other embodiments, a database is based on one or more local computer storage devices.

[0145]

[0155] Provided herein are kits related to the methods, compositions and systems described herein. In some embodiments, the kits may comprise a plurality of recognition elements, a plurality of detection polynucleotides, one or more buffers, one or more reagents, instructions for use, a manual, a protocol, or a combination thereof. The kits may comprise a plurality of recognition elements. The kits may comprise a plurality of detection polynucleotides. The kits may comprise one or more buffers. The kits may comprise one or more reagents. The kits may comprise instructions for use. The kits may comprise a manual. The kits may comprise a protocol.

[0146]

[0156] In some embodiments, a kit may comprise one or more antibody-oligonucleotide conjugates as disclosed herein. In some embodiments, a kit may comprise one or more buffers. In some embodiments, a kit may comprise two or more buffers. In some embodiments, a first buffer of a kit may be configured to promote hybridization. In some embodiments, a second buffer of a kit may be configured to promote de-hybridization, ligation, nucleic acid digestion, storage of a purified molecule. In some embodiments, a kit may comprise one or more reagents. In some embodiments, a kit comprises one or more enzymes. In some embodiments, a kit comprises one or more of a ligase, a DNA polymerase, and an exonuclease. In some embodiments, a kit may comprise instructions for use, a manual, a protocol, or a combination thereof. In some embodiments, a kit may comprise one or more 96 well plates. In some embodiments, one of the 96 well plates of a kit is configured to be assayed by an optical imaging device described herein.

[0147]

[0157] In some embodiments, for an encoded assay, a target protein molecule is detected and associated with a specific barcode that identifies the presence of the target protein molecule. In some embodiments, the additional association of a target protein with a specific barcode in conjunction with a unique code of a recognition element can be used as a proxy for detection and identification of the original target protein molecule from, on or in a sample. In some embodiments, the target protein molecule is an antigen. In some embodiments, the antigen is an immune cell antigen. In some embodiments, the target protein molecule is an intracellular protein. In some embodiments, the target protein molecule is a transmembrane protein. In some embodiments, the target protein molecule is a surface protein. In some embodiments, the target protein molecule in an extracellular protein. In some embodiments, there is no limitation as to what type of protein, or the location of a protein, of interest is to be queried, only that the protein of interest is available to bind to an antibody of an antibody-oligonucleotide conjugate.

[0148]

[0158] In some embodiments, an encoded assay comprises a recognition event in which a target molecule is uniquely recognized by an antibody-oligonucleotide conjugate, and the oligonucleotide of the conjugate is recognized by a recognition element. In some embodiments, the recognition event comprises an antibody-oligonucleotide conjugate wherein the antibody recognizes a target protein or a fraction or variant thereof as a target molecule, and wherein the oligonucleotide comprises a barcode that identifies the antibody to which it is conjugated, thereby identifying the protein to which the antibody is bound. In some embodiments, the oligonucleotide of the conjugate is used as a proxy of the antibody binding to its antigen, where in turn a portion of the sequence of the oligonucleotide is recognized and bound to a recognition element. The recognition element may comprise, among other sequences, a code that is unique to the bound oligonucleotide and therefore the antibody binding event, and therefore the protein target of interest to which the antibody is bound. A detection polynucleotide can be hybridized to the code, or a portion thereof, in an amplification product of the recognition element, and the resulting detectable signal can be imaged, decoded and correlated back to the original target protein molecule.

[0149]

[0159] In some embodiments, an antibody (or a fraction of an antibody) of the antibody- oligonucleotide conjugate recognizes and binds a target protein molecule that is excreted from one or more cells, a protein that is intracellular, a protein that is membrane bound in a cell, or proteins that are in the extracellular matrix surrounding tissues and cells. In some embodiments, the oligonucleotide attached to the antibody comprises one or more barcodes that identify the antibody binding event to the target protein molecule.

[0160] In some embodiments, upon binding of the antibody to its target protein molecule, the oligonucleotide of the antibody-oligonucleotide conjugate is amplified to produce a plurality of amplicons comprising the one or more oligonucleotide associated barcodes. In some embodiments, the oligonucleotide is released from the antibody, for example by enzymatic or chemical means. In some embodiments, the oligonucleotide is used in amplification as it is conjugated to the antibody. In some embodiments, the oligonucleotide is released from the antibody and the released oligonucleotide is used as a template for amplification of the barcodes. For example, the oligonucleotide is attached to the antibody via a linker, wherein the linker comprises a plurality of uracils which are cleavable by one or more enzymes such as uracil- DNA-glycosylases or DNA glycosylase-lyase endonucleases. A skilled artisan will understand the myriad of options for reversibly conjugating an oligonucleotide to a protein such as an antibody. In some embodiments, upon release of the oligonucleotide from the antibody, the oligonucleotide can be used as a template for amplification or hybridized directly to a recognition element that comprises complementary regions to the released oligonucleotide.

[0150]

[0161] In some embodiments, the antibody (or a fraction of an antibody) of the antibody- oligonucleotide conjugate recognizes and binds to its target protein and sequentially or concurrently the oligonucleotide of the antibody-oligonucleotide conjugate hybridizes to its complementary sequences of a recognition element. In some embodiments, the hybridized recognition element is ligated to form a circular recognition element, which can be amplified to generate concatemeric amplification products that comprise the sequence, or a complement thereof, of the oligonucleotide and a code that identifies the oligonucleotide from the antibody- oligonucleotide conjugate. The code can be detected and decoded for final correlation of the presence, or potential absence, of the target protein in or from a sample.

[0151]

[0162] In some embodiments, the amplification of the oligonucleotide comprises polymerase chain reaction, where the amplification reaction results in a plurality of amplicons. In some embodiments, the amplicons are recognized by specifically designed recognition elements, wherein the recognition elements comprise a 5' end and a 3' end, and the ends of each recognition element are complementary to sequences of the amplified oligonucleotides. In some embodiments, the 5' and 3' ends of a recognition element are complementary to all or a portion of the one or more barcode sequences of the amplicons. In some embodiments, the 5' and 3' ends of a recognition element are complementary to all or a portion of the one or more barcode sequences and all or a portion of sequences that flank the one or more barcode sequences. In some embodiments, the 5' and 3' ends of a recognition element recognize and hybridize to an amplicon of the plurality of amplicons. In some embodiments, the recognition ends hybridize to their target sequences on the amplicons either adjacently or non-adjacently. In some embodiments, if hybridization is non-adjacent, a gap fill extension reaction can be performed to extend the 5' end of the recognition until it abuts and is adjacent to the 3' end. Alternatively, in some embodiments, if hybridization is non-adjacent a third oligonucleotide can be inserted and hybridized into the gap and extension, or not, of the third oligonucleotide can be performed.

[0152]

[0163] In some embodiments, after an amplicon hybridizes to its complementary sequences of a recognition element, the 5' and 3' ends of the recognition element are ligated due to their close proximity. If there is no hybridization of an amplicon to the ends of a recognition element, there is no ligation. In some embodiments, ligation of a recognition element thereby provides a circularized recognition element. In some embodiments, a recognition element as provided in this disclosure comprises end sequences that are complementary to one or more barcodes and optionally additional sequences that are present on an oligonucleotide of an antibody- oligonucleotide conjugate.

[0153]

[0164] Amplification of the oligonucleotides from antibody-oligonucleotide conjugates wherein the antibody is bound to its target protein, may serve to amplify the regions that are targeted by recognition elements. In some embodiments, the one or more barcodes is unique for each antibody-oligonucleotide conjugate. In some embodiments, the one or more barcodes is not unique for each antibody-oligonucleotide conjugate. For example, each oligonucleotide of an antibody-oligonucleotide conjugate comprises one or more barcodes that is specific in its identification of the antibody to which it is conjugated, whereas in other instances the one or more barcodes is duplicated on one or more other oligonucleotides of other antibody- oligonucleotide conjugates. However, even if the one or more barcodes is shared between two or more oligonucleotides, downstream assay methods can be used to uniquely identify which oligonucleotide is associated with a particular antibody which recognizes and binds to a particular target protein, for example by detecting the code of the recognition element of one which is different from the code of the other recognition element.

[0154]

[0165] A recognition element of the present disclosure is illustrated in an example recognition element shown in Fig. 1. In some embodiments, a target molecule nucleic acid sequence, in this disclosure an amplicon generated from an oligonucleotide of an antibody-oligonucleotide conjugate or a released oligonucleotide from the antibody-oligonucleotide conjugate, is recognized and hybridized to the 5' and 3' ends of a recognition element. In some embodiments, hybridization serves to bring the two ends of a recognition element together, which is nascently in a linear configuration. In some embodiments, hybridization of the target sequences to its complementary ends of the recognition element results in a padlock probe configuration for the recognition element.

[0166] A recognition element may further comprise an amplification primer binding site and a code, at minimum. In some embodiments, the amplification primer binding site is universal in nature and is the same for each recognition element regardless of target molecule identification. In other embodiments, the amplification primer binding site is not universal and is different for one or more recognition elements. In some embodiments, a recognition element further comprises a code that is unique for each recognition element. In some embodiments, a code or a portion of a code can serve as an amplification primer binding site. In some embodiments, the code in each recognition element is used to uniquely identify, and is correlated with, the target molecule of interest. As such, while the one or more barcodes of an oligonucleotide of an antibody-oligonucleotide conjugate may be the same, each of the oligonucleotides can be identified and correlated back to the original target protein molecule of interest due to the unique code of a recognition element to which it is associated with.

[0155]

[0167] In some embodiments, after ligation and circularization of a recognition element that had hybridized to its complementary target sequence of the oligonucleotide derived amplicons of the oligonucleotide itself, the circularized recognition element can be amplified. In some embodiments, amplification of a circularized recognition element is performed by hybridizing an amplification primer that is complementary to the amplification primer binding site in the recognition element. In some embodiments, amplification comprises extension by a DNA polymerase. In some embodiments, amplification is rolling circle amplification, multiple strand displacement amplification, or other amplification method that generates an amplification product comprising multiple copies of the circularized recognition element.

[0156]

[0168] In some embodiments, the concatemeric amplification product is decoded by detecting and deciphering the code as a proxy for identifying the presence of the original target analyte, for example a target protein that was first detected by binding of the protein to an antibody of an antibody-oligonucleotide conjugate. In some embodiments, detection of the presence of the code can be performed by a variety of methods. In some embodiments, detection is performed by hybridizing, in one or more detection cycles, one or more fluorescently labeled detection polynucleotides which specifically hybridize to code sequences of a concatemeric amplification product and detecting the signals from the hybridization events. In some embodiments, detection is performed by sequencing the concatemeric amplification products, for example by sequence by synthesis, sequence by hybridization, single molecule sequencing, nanopore sequencing, and the like. In some embodiments, the decoding of the detected signals, either from hybridization of detection polynucleotides or by sequencing, is performed using a soft decoding probability assessment based on the signals detected. The codes may be error corrected and thus easy to distinguish one code from another code, as such a code can be detected at low abundance, in the presence of a high level of background and in the presence of many other codes from many other recognition elements that may be present in a multiplexed encoded assay.

[0157]

[0169] In some embodiments, the operations of hybridization of a recognition element to a target sequence, ligation of the recognition element, extension amplification of the recognition element, and detection of the amplified codes may occur sequentially, or combinations of the operations, including all of the operations, may occur simultaneously as a single combined operation. The signals captured during detection can be subsequently decoded in identifying the presence, or absence, of a target protein in or from a sample.

[0158]

[0170] As such, methods and assays described herein find utility for multiplexed and multiomic assays as every recognition element comprises a unique code that can be correlated to a target molecule of interest and decoding is therefore unique and can be correlated and tied back to each target molecule. Multiple parallel workflows that are target molecule dependent can be decoded simultaneously in a single platform. Parallel assay workflows can be merged into a single workflow, where multiple targets and target-types (e.g., nucleic acids, polypeptides) may be decoded simultaneously in a single workflow and also read simultaneously within the same readout platform. With respect to the present disclosure, the present methods allow for detection and identification of multiple cell related target protein molecules in a single assay.

[0159]

[0171] The disclosure provides assays for multiplexed detection of target molecules. In various embodiments, the assays provide a readout that can be measured alongside the readout of other molecular assays that may be performed in parallel, thereby enabling a multiomic platform for the analysis of different analytes in a sample. The target may be any biomolecule (e.g., carbohydrate, protein, nucleic acid, or small molecule, or the like). For example, protein targets that identify the presence of one or more cellular proteins from a sample can be performed in parallel with a nucleic acid target assay for identifying the presence of mRNA or gDNA from the same sample. In some embodiments, the detection of both proteins and nucleic acids can be performed concurrently or simultaneously in one assay. In some embodiments, the detection of proteins and nucleic acids can be performed in a step like manner, for example first protein detection can be performed and then nucleic acid detection on the same sample can be performed, or vice versa. In some embodiments, a sample can be divided into two separate aliquots, wherein the first aliquot is subjected to target protein detection following the methods described herein and the second aliquot can be subjected to target nucleic acid detection following the methods described herein. In some embodiments, the methods described herein can be leveraged for detecting target proteins and associated target nucleic acids to provide a sample analysis comprising the presence or absence of a protein of interest and the presence of a nucleic acid associated with a protein, thereby reporting a multiomic assessment of a sample.

[0172] An assay for detecting target proteins may include: (i) a capture event, in which a target protein is uniquely recognized and bound by a capture agent such as an antibody, antibody fragment, aptamer, etc. to form a capture agent-target complex such as an antibody oligonucleotide complex wherein the antibody is bound to its target protein; (ii) a recognition event, in which the oligonucleotide which comprises one or more barcodes that identify the antibody to which it is associated and thereby the target protein which binds the antibody binds, or a complement thereof or amplicons thereof, is uniquely recognized by and hybridized to a recognition element comprising a code; (iii) a transformation event, in which the hybridized and circularized recognition element is ligated and subsequently amplified to generate concatemeric amplification products; and (iv) a detection and decoding event, that uses the code of the recognition element as a proxy for identifying the target protein molecule by recognizing and determining the presence of the code in combination with the antibody specific barcode in the oligonucleotide of the antibody-oligonucleotide conjugate.

[0160]

[0173] In some embodiments, assays for identifying the presence or a target protein molecule from or in a sample comprises a capture agent such as an antibody or fragment thereof that is specific for a target protein of interest. In one example, the antibody (or fragment thereof) may, for example, be used in an immuno-detection process, such as a modified enzyme-linked immunosorbent assay (ELISA). In some embodiments, the capture agent may be an aptamer (e.g., an oligonucleotide or peptide sequence) that is specific for a target protein molecule of interest. In some embodiments, the capture agent may be a synthetic (or semi-synthetic) molecule such as one that includes unnatural amino acids or bases. The number of capture agents may be increased or decreased depending on the desired plexity of the assay.

[0161]

[0174] In some embodiments, a capture agent of the present disclosure further comprises an oligonucleotide, to generate a capture agent complex. In some embodiments, an oligonucleotide of the present disclosure comprises one or more barcode sequences, wherein the one or more barcode sequences identifies the capture agent, such as an antibody, to which it is bound. An oligonucleotide of the present disclosure can further comprise one or more common, or universal sequences, that flank the one or more barcodes. The common sequences of the oligonucleotide include, but are not limited to, amplification primer binding sites, capture sites, restriction site, unique molecular identifiers, cleavage sites, or a combination thereof. As such, the oligonucleotide of a capture agent complex, for example an antibody-oligonucleotide conjugate, can be used to link the specificity of the capture agent, the antibody, to the target protein. Therefore, an oligonucleotide of an antibody-oligonucleotide conjugate can be used in a secondary way to identify the presence of the target protein to which the antibody is bound.

[0175] Additional methods for identifying a protein and / or nucleic acid in a sample can be found in WO2023 / 096674A1, which is incorporated herein by reference in its entirety.

[0162]

[0176] Fig. 8 is an example of a capture agent complex for use in assays of the present disclosure. In the example shown in Fig. 8, the capture agent is an antibody, and the oligonucleotide is linked to that antibody, resulting in an antibody-oligonucleotide conjugate. The linkage can be covalent or non-covalent. In some embodiments, the linkage is a cleavable linkage, either via chemical cleavage, enzymatic cleavage, or photocleavage. The oligonucleotide in this example comprises two functional sequences. In some embodiments, the functional sequences are universal sequences. The universal sequences could be the same, or they could be different one from the other. For example, one of the universal sequences could be an amplification primer binding site, for example for use in initiating an extension reaction, whereas the other universal sequence could be a capture sequence, or a cleavage site, etc. Another example is that both universal sequences serve as amplification primer binding sites for PCR amplification. The sequences are universal because they are found in other oligonucleotides that are bound to additional antibodies that target different target proteins of interest. Alternatively, each of the functional sites could be different from one oligonucleotide to the next, depending on the needs of the assay. Additionally, an oligonucleotide that is linked to a capture agent comprises a unique sequence that identifies its capture partner. In this example, the unique sequence is called a barcode. The barcode serves to indirectly identify the presence of a target protein of interest as it correlates to the capture agent which binds the target protein.

[0163]

[0177] In some embodiments, an antibody-oligonucleotide conjugate is added to a sample and the antibody recognizes and binds its target protein. The binding can be done in solution, on a solid support, on tissues, cells, lysates, and the like. After unbound antibody-oligonucleotide conjugates are removed the oligonucleotide can be optionally cleaved from the antibody and released. The oligonucleotide that is released can be amplified to increase the possibility of the oligonucleotide hybridizing to a recognition element, or it can be released oligonucleotide can be captured directly by a recognition element. In some embodiments, the oligonucleotide is amplified prior to release. Alternatively, in some embodiments the oligonucleotide is not released from the antibody, but instead the barcoded region of the oligonucleotide is amplified directly from the antibody oligonucleotide complex using primers that are complementary to sequences that flank the barcode sequence, thereby generating barcoded amplicons that are captured by a recognition element. Fig. 9 is an example of an oligonucleotide amplicon that includes two barcode sequences that are adjacently hybridized to a recognition element. In this example shown in Fig. 9, the 5' and 3' ends of the recognition element are complementary to one of the two barcodes and additionally a portion of one of the flanking amplification binding site sequences that were used for amplification. The oligonucleotide amplicon / recognition element complex at this point includes a code which uniquely identifies the target oligonucleotide, which in turn uniquely identifies the antibody to which it was associated, the combination of which uniquely identifies the original target protein of interest bound to the capture agent complex.

[0164]

[0178] As such, Fig. 10 demonstrates an example of a proteomics workflow of the present disclosure. Following recognition and binding of a target protein of interest with an antibody - oligonucleotide conjugate, excess non-bound antibody-oligonucleotide conjugates are washed away, and the oligonucleotide of the conjugate is optionally amplified, wherein the amplicons comprise the barcode(s) present on the oligonucleotide. The amplicons serve as a proxy of the antibody binding to its target protein due to the inclusion of the barcode(s), while at the same time the barcodes and potentially adjacent sequences serve as target nucleic acids for hybridizing to complementary sequences present on a recognition element. Successful hybridization of the target nucleic acids to the recognition elements enables ligation of the 5’ phosphorylated end of the recognition element to the adjacent 3’ end, thereby circularizing the recognition element. The pool of circularized recognition elements is exposed to exonuclease digestion to remove unligated recognition elements and target nucleic acids. The resulting pool of circularized recognition elements is transferred to custom 96 well plates, amplified into single-stranded concatemers and detected and decoded on a fluorescent reader.

[0165]

[0179] As disclosed herein, the target molecules of interest are protein targets. In some embodiments, the protein targets of interest are exemplified by immune cell associated proteins. In some embodiments, immune cell associated proteins include extracellular proteins excreted from immune cells. In some embodiments, immune cell associated proteins include intracellular proteins. In some embodiments, immune cell associated proteins include surface bound proteins, in some embodiments immune cell associated proteins include transmembrane bound proteins.

[0166]

[0180] In some embodiments, the ability of the disclosed assay to detect and identify the presence of cell related proteins is demonstrated and exemplified by detecting and identifying the presence of a subset of proteins associated with T lymphocytes. In some embodiments, the target proteins include T lymphocyte excreted proteins including, but not limited to, one or more of IFNg, IL-10, IL-2, IL-6, LAG-3, PD-1, PD-L1, and TNFa. In some embodiments, the target proteins include intracellular proteins in T lymphocytes, including but not limited to, one or more of IFNg, IL-2, IL-10, IL-6 and TNFa. In some embodiments, the target proteins include surface bound proteins in T lymphocytes, including but not limited to, one or more of, CD25, CD3, CD4, CD8, CTLA-4, LAG-3, PD-1 and PD-L1. In some embodiments, an assay for target proteins associated with T lymphocytes as disclosed herein includes a combination of two or more of extracellular, intracellular and surface bound proteins.

[0181] Provided herein are kits related to the methods and systems described herein. In some embodiments, the kits may comprise a plurality of recognition elements, a plurality of detection polynucleotides, one or more buffers, one or more reagents, instructions for use, a manual, a protocol, or a combination thereof. In some embodiments, a kit may comprise one or more antibody-oligonucleotide conjugates.

[0167] In some embodiments, a kit may comprise one or more buffers. In some embodiments, a kit may comprise two buffers. In some embodiments, a first buffer of a kit may be configured to promote hybridization. In some embodiments, a second buffer of a kit may be configured to promote dehybridization. In some embodiments, a kit may comprise one or more reagents. In some embodiments, a kit comprises one or more enzymes. In some embodiments, a kit comprises one or more of a ligase, a DNA polymerase, and an exonuclease. In some embodiments, a kit may comprise instructions for use, a manual, a protocol, or a combination thereof. In some embodiments, a kit comprises one or more 96 well plates. In some embodiments, a kit comprises two 96 well plates, or three 96 well plates or more. In some embodiments, one of the 96 well plates of a kit is configured to be assayed by an optical imaging device described herein.

[0168] EXAMPLES

[0169] Example 1- Use of synthetic oligonucleotides for determining assay efficacy

[0170]

[0182] This example describes a proof of concept workflow using mock synthetic target oligonucleotide constructs as test samples for proteins, in this example proteins that are typically associated with T lymphocytes, including intracellular / surface bound proteins and extracellular proteins. The synthetic target oligonucleotides included two pools, one pool mimicked extracellular protein targets IFNg, IL-10, IL-2 #2, IL-6, LAG-3 #2, PD-1, PD-L1 and TNFa. The intracellular / surface bound pool mimicked the protein targets CD25, CD3, CD4, CD8, IFNg, LAG-3 #1 IL- 10, IL-2 #1, IL-6 and TNFa.

[0171]

[0183] Two controls were run in tandem with the synthetic target oligonucleotides. The first control is a standard curve that were generated for the 1) extracellular protein panel and the 2) intracellular / surface bound protein panel. The standard curves are prepared by serial dilution of a pool of equimolar concentration of all synthetic target oligonucleotides specific for each protein target group (e.g., eight for the extracellular target panel and 13 for the intracellular / surface bound target panel). Each synthetic target oligonucleotide is quantified using one of the two standard curves and the standard curves are also expected to help account for any differences in workflow operations, such as differences in ligation, amplification, and detection inefficiencies for each synthetic target oligonucleotide.

[0184] Standard curve serial dilutions are made starting with a 100 nM stock solution of equimolar amounts of synthetic target oligonucleotides associated with 1) the extracellular targets, and separately for the 2) intracellular / surface bound targets. Serial dilutions are performed wherein the final concentrations added to the recognition elements for capture and ligation are 35 pM, 12.5 pM, 4.46 pM, 1.59 pM, 0.57 pM, 0.203 pM, 0.073 pM and 0.026 pM. The serial dilutions are put through the same workflow as the synthetic test samples, in duplicate (n=2).

[0172]

[0185] The second control set is generated with synthetic oligonucleotide targets and their recognition elements designed with specific codes and specific target recognition element regions directed to the specific synthetic targets. A second set of controls is included to provide decoding signal diversity for optimizing color signal registration of the concatemers by the RAPTOR instrument (Pleno, Inc.) during detection as well as for use in normalizing the counts for assay wells during decoding. The second set of controls is also used as quality indicators for the enzymatic operations in the workflow. The second set of controls is exposed to the same workflow as the synthetic test samples, except that during detection all four different fluorescently labeled detection polynucleotide complexes are present in every query cycle.

[0173]

[0186] A negative sample control, sample without the synthetic oligonucleotides, is also run concurrently with the other samples. There is one negative control for each of the protein targets.

[0174]

[0187] Synthetic single stranded target oligonucleotides are generated that are identical to sequences of the oligonucleotides that would be conjugated to antibodies for identifying the presence of a particular protein associated with a T lymphocyte. As such, the synthetic target oligonucleotides provide a mechanism for determining whether the recognition elements could recognize and hybridize with specificity to the target oligonucleotide sequences that would be used in antibody oligonucleotide complexes for protein identification.

[0175]

[0188] For this proof of concept example, amplification of an oligonucleotide of the antibody- oligonucleotide conjugate is mimicked by hybridizing two single stranded synthetic target oligonucleotides to generate double stranded DNA “amplicons”. This was done for each of the synthetic target oligonucleotides for the extracellular and intracellular / surface bound panels. At this stage, the dsDNA synthetic target oligonucleotides for the two panels are kept in separate tubes. The two panels are initially separated as several of the barcode combinations associated with the antibodies were duplicated between the two panels.

[0176]

[0189] Recognition elements are designed for this proof of concept experiment, wherein the 5’ recognition element region and the 3’ recognition element region were designed as complements of the two different barcodes and portions of their flanking common regions, respectively, of an oligonucleotide associated with a specific antibody-oligonucleotide conjugate being tested. Other sequences of the recognition were as previously described, for example each recognition element includes a primer amplification binding site and a code, wherein each code is unique to each recognition element.

[0177]

[0190] In a 96 well plate, the standards curves, controls and test samples are added to separate wells, all samples are duplicated by generating two sample 96 well plates. Two master mixes of recognition elements, ligase enzyme (e.g., Ampligase or other thermostable ligase) and associated buffers are added to each well, one for either the extracellular or the intracellular / surface bound targets, controls and standard curve. Hybridization of the recognition elements to their targets, and subsequent ligation of the hybridized recognition elements is performed in a thermocycler; three cycles of 95°C / lmin, ramp down to 61°C, 61°C / 20min, with a hold 4°C after cycling.

[0178]

[0191] After hybridization and ligation, the samples are treated with an exonuclease mix (e.g., a mixture of Exo I, Exo III, Lambda exo or other exonucleases) to remove linear nucleic acids. As such, ligated and circularized recognition elements are not a substrate for exonuclease digestion and they remain in solution. The exonuclease digestion reaction is performed at 37°C / 30 min, 80°C / 30min and hold at 4°C.

[0179]

[0192] Post exonuclease digestion clean up, the extracellular and intracellular / surface bound wells are combined, as are the standard curves and the controls, all in separate tubes per like kind, and an amplification master mix (e.g., EquiPhi polymerase or other RCA capable polymerase) is added to the combined circularized recognition elements. While there are a few barcode combinations that are repeated for the oligonucleotides associated with extracellular and intracellular / surface bound antibody conjugates, each recognition element code is unique to each target protein. The circularized recognition elements and amplification mix samples are reallocated into a 96 well, optically designed plate for the amplification reaction to proceed, 42°C / 2hrs. The amplification reaction is stopped, samples washed, and the 96 well plate with the amplification products are sealed and stored at 4°C prior to detection.

[0180]

[0193] Detection is performed by multiple cycles of hybridization and imaging with detection polynucleotides that are complementary to code sequences in the concatemeric amplification products and are fluorescently labeled with one of AZ532, AZ568, AZ647 or AZ680 fluorescent moiety. Imaging is performed by collecting fluorescence by filtering for either green fluorescent emission (excitation at 530nm for AZ 532 and AZ568) or red fluorescent emission (excitation at 639 for AZ647 and AZ680). The different cycles of hybridization and imaging provide a pattern that is characteristic of the code present on any given concatemeric amplification product, which in turn correlates back to the barcoded oligonucleotide that is bound to the target protein through the hybridization of the barcoded oligonucleotide of the target protein bound antibody- oligonucleotide conjugate to the 5’ and 3’ complementary sequences present on a coded recognition element.

[0181]

[0194] An example of a standard curve serial dilution graph generated from data practicing Example 1 is seen in Fig. 11. The standard curve is based on serial dilution of a pool of all targets at equimolar concentration. The synthetic oligonucleotides are successful in hybridizing to their complementary sequences on recognition elements, as evidenced by the generation of concatemeric amplification products for each synthetic oligonucleotide as seen in Fig. 12. Data representing 15 total synthetic targets being assayed, with three targets at each concentration, showed that, for this experiment, the calculated concentration of targeted protein was highly concordant with target input concentration (R2>0.98). The calculated concentrations were highly accurate, with <20% error, and the assay was highly reproducible with inter-assay and intraassay CVs at <15% and <10%, respectively. Further, detection and decoding on the RAPTOR instrument demonstrated a six log dynamic range. Negative controls were easily distinguishable from the lowest concentration in the standard curves, and calculated concentrations of the negative controls associated with the target protein synthetic oligonucleotides were well below the lower limits of detection. As such, the experiment was successful in determining whether specially designed coded recognition elements could, with a high degree of sensitivity and specificity, hybridize with synthetic oligonucleotides that served as proxies of oligonucleotides from potential antibody-oligonucleotide conjugates for use in detecting protein targets present in a sample.

[0182] Example 2-Determining the presence of T lymphocyte related proteins in a sample

[0183]

[0195] Antibodies are conjugated to oligonucleotides that include at least one barcode and at least two common regions. For additional correlation between an antibody and its conjugated oligonucleotide, two or more barcodes can be included in the oligonucleotide (Figs. 8 and 9).

[0184]

[0196] Table 1 lists target immune proteins used for evaluating the methods, as applicable their location in a T cell, and exemplary antibodies that can be used for target protein recognition and binding. Table 1-T lymphocyte and immune system associated protein targets

[0185]

[0197] In this example, two separate assays are set up due to barcodes that are the same for antibodies that overlap and can identify extracellular and intracellular / surface bound proteins. Antibodies listed are on offer from BioLegend, however any antibody from any source can be used as long as it meets the defined characteristics of the assay.

[0186]

[0198] The antibodies are conjugated to their respective oligonucleotide following established protocols. For example, AbCam offers an Oligonucleotide Conjugation Kit (ab218260 Oligonucleotide Conjugation kit) for generating antibody-oligonucleotide conjugates. Briefly, single stranded oligonucleotides are generated, wherein each oligonucleotide sequence included one or more barcodes that are used to identify the antibody it is conjugated to, and thereby the target protein that is recognized and binds the antibody, and at least two common sequences that flank the one or more barcodes. For example, if an oligonucleotide includes a common sequence that is an amplification primer binding site sequence of around 20 nucleotides (nt) and the second common sequence includes a capture sequence or other functional sequence of around 20 nt, and a barcode is around 8 nt, then the synthetic oligonucleotide that identifies an antibody, and is bound to the antibody, is around 48 nt long. Other oligonucleotide constructs are also contemplated as described herein. Regardless of oligonucleotide length, the user guide associated with the AbCam kit states a terminal amine group is added during oligonucleotide synthesis. Oligonucleotides that are 5’ aminated are typically more efficient when used for conjugation. The 5’ aminated oligonucleotide is purified. The antibodies are purified as well prior to conjugation with their oligonucleotides.

[0199] The oligonucleotides and antibodies are activated for 30 min. at room temperature and desalted. Depending on the desired antibody: oligonucleotide ratio, different amount of the desalted antibody and desalted oligonucleotide are added to a conjugation reaction. The conjugation reactions are incubated at room temperature for at least one hour, or overnight as needed. If desired, the resulting antibody-oligonucleotide conjugates can be further purified to remove any unconjugated antibodies and oligonucleotides. The antibody-oligonucleotide conjugates can be analyzed for expected conjugate size via gel electrophoresis such as SDS- PAGE gel.

[0187]

[0200] The antibody-oligonucleotide conjugates can be biotinylated, wherein the antibody is biotinylated. This can be an important operation especially when extracellular proteins are to be identified. As the extracellular target proteins are not cell associated, a mechanism for binding them in solution via the antibody-oligonucleotide conjugate and then capturing the antibody- oligonucleotide conjugate. Streptavidin coated beads can then be used to capture the biotinylated antibody-oligonucleotide conjugate that has bound to its target protein.

[0188]

[0201] A skilled artisan will understand that there are multiple ways to attach single stranded oligonucleotides to antibodies. The use of a commercial kit as described is exemplary and does not limit the present disclosure.

[0189]

[0202] Once the antibody-oligonucleotide conjugates are generated and quality control analyzed, they can be used to identify their specific protein of interest.

[0190]

[0203] Tissues, cells or other biological material can be used as a primary sample for use in assays of the present example. For example, for identification of intracellular or surface bound proteins, a tissue biopsy, tissue section, buffy coat suspension, sorted T lymphocyte suspension, or other type of biological material can be placed in tubes, wells, or other type of chamber for protein detection and assay. For extracellular proteins for immune cells, plasma or serum can be harvested from a coagulated or anti -coagulated blood sample. For this example, a sample from which immune cell proteins, in the case T lymphocyte proteins, are to be detected is placed in wells of a 96 well plate. A blood sample taken from a subject with a Vacutainer tube that includes EDTA is spun down for 10 min. at 1000 x g and the buffy coat is removed, washed, resuspended is isotonic saline and aliquoted into different wells of a 96 well plate for intracellular / surface bound protein detection. For the extracellular proteins, a sample of the plasma from the EDTA Vacutainer tube is removed after centrifugation and aliquoted into the wells. If the assay is not going to be performed immediately, plasma samples can be stored at - 20°C until ready to use. As the extracellular proteins are excreted from the cells, it is important to get as much of what might be included in the extracellular matrix as possible to increase the chances of detecting these proteins.

[0204] Protein targets for detection associated with T lymphocytes are identified in Table 1. A standard curve and controls are designed and generated using the general directions as found in Example 1 and are aliquoted into wells of the 96 well plate. The cells in the intracellular / surface bound wells are permeabilized to allow for entry of the antibody-oligonucleotide conjugates into the cell. A negative control for each plate where no target proteins are included, is also present on each plate.

[0191]

[0205] The pool of extracellular or intracellular / surface protein targeted antibody- oligonucleotide conjugates are added to the wells of the appropriate test plate, in this example two plates for the two different protein locations, their standard curves and controls. The antibodies are allowed to bind to their target proteins. Following binding, the wells can be washed to remove any unbound antibody-oligonucleotide conjugates, followed by amplification of the oligonucleotide barcoded region, wherein PCR uses the two common areas which are universal for all oligonucleotides as the PCR primer binding sites, thereby generating a plurality of amplicons from the oligonucleotides of the bound antibody-oligonucleotide conjugates, wherein the amplicons include the common sequences and the barcodes that are associated with the antibody from which they were conjugated. For extracellular protein detection, biotinylated antibody-oligonucleotide conjugates which have bound to their target protein can be captured by streptavidin coated beads and washed, followed by amplification of the oligonucleotide barcoded region as previously described. The amplicons generated from both pools of target proteins can be combined and aliquoted into wells of a 96 well plate to which are added coded recognition elements comprising 5’ and 3’ regions that are complementary to a specific barcode sequence and adjacent common sequences, an amplification primer sequence and a code that is unique to the recognition element and hence the oligonucleotide barcode that is associated with an antibody to a particular target protein. The coded recognition elements are allowed to hybridize to their target sequences, the hybridized recognition elements are circularized via ligation, and linear nucleic acids are removed by exonuclease digestion, for example using the methods as described in Example 1.

[0192]

[0206] As with Example 1, the test wells from the two plates can now be concatenated into one combined test sample as each recognition element includes a unique code, wherein any barcodes that are shared can now be uniquely separated and used to identify the original protein target of interest. The pooled, circularized recognition elements are aliquoted into wells of a 96 well, optically enhanced, plate. Rolling circle amplification is performed for all of the circularized recognition elements, including test sample, standard curves and controls, thereby generating concatemers of the recognition elements. The concatemeric recognition elements are detected and decoded by the RAPTOR instrument and data reported out with regards to the presence, and concentration, of a targeted protein.

[0193] Example 3- Determination of level of detection of barcoded oligonucleotides

[0194]

[0207] This experiment was performed to evaluate what the lower limit of detection might be for the barcoded oligonucleotides that would be linked to an antibody of an antibody - oligonucleotide conjugate for protein detection.

[0195]

[0208] A pool of 100 nM equimolar amounts of four synthetic barcoded oligonucleotides which mimic oligonucleotides that would be conjugated to an antibody for protein detection are generated, and a titration of the pool is created; 0.0001 pM, 0.01 pM, 0.1 pM and 1 pM. A negative control (NTC) with no barcoded oligonucleotides is used to determine the background of the assay. Aliquots of the test titration samples and negative controls are pipetted into wells of a multi-well plate. A master mix is created that includes 1 nM of each recognition element for each of the synthetic oligonucleotide targets, Ampligase, ligase buffer, water and a DNA crowding agent, and 20 pL of the master mix is added to each test and control reaction for a total volume of 50 pL. The hybridization and ligation reactions are carried out at 95°C for 1 min., 61°C for 20 min., for three cycles, then a hold at 4°C.

[0196]

[0209] After the hybridization and ligation cycling, the reactions are exonuclease treated to remove any single stranded recognition elements and barcoded oligonucleotides. To 20 pl of the hybridization and ligation reactions is added 20 pL of a digestion master mix comprising exonuclease I, exonuclease III, digestion buffer and water. The digestion is performed at 37°C for 20 min. followed by enzyme inactivation at 95°C for 5 min, after which the digestion reactions are held at 4°C.

[0197]

[0210] Aliquots of the exonuclease reactions (15 pL) are added to a new well plate and 35 pL of an amplification mix is added, the amplification mix comprises 10 nM of an amplification primer, 1 mM dNTPs, DTT, EquiPhi DNA polymerase, buffer and water. The amplification reactions are incubated at 42°C for 2 hrs, washed with TE several times, the TE is added to the amplification reactions and stored at 4°C.

[0198]

[0211] Detection of the codes of the recognition elements and decoded is performed as in Example 1.

[0199]

[0212] FIGs. 13A-13B show data reporting lower level of detection, LLoD for the synthetic barcoded oligonucleotide mimics. FIG. 13A shows a linear detection range from fM to pM, with an R2=0.99. FIG. 13B expands on the lower limit from FIG. 13A (box), demonstrating that the LLoD was 20 standard deviations above the negative control (NTC) and an LLoD of 1 fM.

[0213] The experiment demonstrates that the synthetic barcoded oligonucleotides that were generated to mimic what would be linked to an antibody for protein detection can be detected over a large range, with high sensitivity of detection of 1 fM for the lower limit of detection.

[0200] Example 4-Effect of antibodies and cellular components on oligonucleotide detection

[0201]

[0214] This experiment was performed to determine if an antibody conjugated to an oligonucleotide would impact the hybridization of the oligonucleotide to a recognition element and subsequent detection of the oligonucleotide as a proxy for the protein target of interest.

[0202]

[0215] Pools of equimolar amounts of the surface bound targeted antibody-oligonucleotide conjugates and the intracellular targeted antibody-oligonucleotide conjugates are generated as listed in Table 2. The oligonucleotides that are conjugated to an antibody are also assayed separately, where they represent the free barcoded oligonucleotides as references in Fig. 14A. The free barcoded oligonucleotides are pooled together, those that would identify surface bound proteins in one pool and those that would identify intracellular proteins in a second pool. The antibody-oligonucleotide conjugates are provided by BioLegend.

[0203] Table 2-Protein targets for antibody-oligonucleotide complexes

[0204]

[0216] A dilution series is titrated for each pool for generating standard curves for the comparison study. An aliquot of each pool is pipetted into wells of a well plate. To the standard curve samples, a hybridization and ligation master mix is added to a total volume of 20 pL, the master mix comprises 1 nM of the targeted recognition elements for each pool, Ampligase, buffer and water. The hybridization and ligation reactions occur simultaneously for three cycles of 95°C for 1 min and 20 min. incubation at 61 °C, followed by placing the reactions at 4°C.

[0205]

[0217] After the hybridization and ligation cycles, the reactions are treated with an exonuclease mix to remove any remaining single stranded recognition elements or single stranded targets. To each reaction is added an exonuclease digestion master mix comprising Exonuclease I, Exonuclease III, buffer, and water for a total volume of 40 pL. The digestion reaction is performed at 37°C for 30 min. followed by enzyme inactivation at 95°C for 5 min. and stored at 4°C.

[0206]

[0218] The exonuclease treated reactions are transferred to wells of a new welled plate and the samples are amplified by a master mix comprising 10 nM amplification primer, EquiPhi DNA polymerase, EquiPhi buffer, 1 pM dNTPs, reducing agent and water to a total volume of added 50 pL. Amplification is performed for 2 hrs at 42°C, washed several times with TE and stored at 4°C in TE. Detection and decoding is performed as in Example 1.

[0207]

[0219] Data shows that there was no detectable effect of the antibody of the antibody - oligonucleotide conjugate on the oligonucleotide hybridization to its targeted recognition element. As seen in Fig. 14A, the oligonucleotide linked to the antibody hybridized to its target recognition element in the same manner as the free barcoded oligonucleotide that was not linked to an antibody. As such, the antibody conjugated to an oligonucleotide does not impact the oligonucleotide hybridization to its target recognition element.

[0208] Example 5- Effect of background materials on free barcoded oligonucleotide hybridization

[0209]

[0220] Based on the results from Example 4, free barcoded oligonucleotides are used as a proxy for the oligonucleotides that are linked to antibodies for protein target detection. Experiments are performed to evaluate whether cellular matrix materials found in a cell lysate might impact barcoded oligonucleotide hybridization to targeted recognition elements.

[0210]

[0221] For the first experiment, a sample comprising DNA, RNA matrix components from a non-permeabilized PBMC sample are evaluated. Briefly, PBMCs are isolated from plasma. The cells are washed, incubated in a buffer associated with an antibody (e.g., Fc blocked) and washed again. A portion of the washed cells are lysed with proteinase K and nucleic acids are isolated. The isolated sample therefore comprises DNA, RNA, and matrix components.

[0211]

[0222] For the second experiment, a sample comprising the cellular nucleic acids including matrix components from permeabilized PBMCs is generated. Briefly, after washing of the PBMCs, a portion of the cells are permeabilized and fixated. The permeabilized and fixated cells are washed and lysed with proteinase K, providing lysed samples comprising nucleic acids and cellular fixation matrix components.

[0212]

[0223] The non-permeabilized and permeabilized and fixated samples are mixed with free barcoded oligonucleotides to simulate the oligonucleotide that would be linked to a protein targeted antibody.

[0213]

[0224] Fig. 14B shows results of the sample lysate components mixed with free barcoded oligonucleotides. The data in Fig. 14B demonstrates that the components in the non- permeabilized samples (DNA / RNA) and permeabilized samples (DNA / RNA / cell fixation) appeared to have no impact on the hybridization of the free barcoded oligonucleotides to their target recognition elements.

[0214]

[0225] As such, the data suggest that lysate matrix components do not impact the hybridization of a proxy free barcoded oligonucleotide to its targeted recognition element.

[0215] Example 6- Protein detection from peripheral blood mononuclear cells

[0216]

[0226] This experiment was performed to directly detect proteins from cells using the methods described herein, the protein targets are those from T-cells. As outlined in Fig. 15, a whole blood sample comprising plasma is spun down and the peripheral mononuclear blood cells, PMBCs, layer, or buffy coat, is isolated from the other blood components. The PMBCs are bifurcated into a portion that is stimulated by addition of phorbol myristate acetate and Brefeldin A, and a portion that is unstimulated. Both stimulated and unstimulated cells are assayed in the same manner.

[0217]

[0227] To each of the PMBC portions is added a pool of antibody-oligo conjugates for binding to targeted surface bound proteins (see Table 2). The antibodies of the antibody-oligonucleotide conjugates are allowed to bind to their targets and the cells are washed. The washed cells are permeabilized and the pool of antibody-oligonucleotide conjugates that recognize and bind to intracellular proteins is added (see Table 2). The intracellular targeted antibody-oligonucleotide conjugates are allowed to bind to their targets and the cells are washed. The washed cells are lysed by the addition of Proteinase K and the total nucleic acid portion is isolated, and a portion thereof added to reactions as outlined in Fig. 15 and as previously described for hybridization, ligation, amplification, detection and decoding.

[0218]

[0228] The graph in Fig. 16A demonstrates that the methods described herein can detect and quantify surface bound and intracellular proteins from PBMCs, and further Fig. 16B demonstrates that the methods described herein are responsive in detecting differences in protein target amounts when cells are stimulated compared to unstimulated cells, for both surface bound proteins and intracellular proteins.

[0219]

[0229] As such, the data demonstrate that the methods described herein are able to detect and quantify protein targets on and in cells, in this example targets associated with peripheral mononuclear blood cell proteins which were used as a test system for method evaluation.

[0220]

[0230] While certain examples of methods and systems have been shown and disclosed herein, one of skill in the art will realize that these are provided by way of example only and not intended to be limiting within the specification. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the scope disclosed herein. Furthermore, it should be understood that all aspects of the disclosed methods and systems are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables and the description is intended to include such alternatives, modifications, variations or equivalents.

Claims

CLAIMSWhat is claimed is:

1. A method for identifying a presence of a protein of interest from a sample, comprising: a) binding an antibody-oligonucleotide conjugate to the protein of interest from the sample, wherein the antibody-oligonucleotide conjugate comprises an antibody that recognizes and binds to the protein of interest and an oligonucleotide that is attached to the antibody, wherein the oligonucleotide comprises at least two functional sequences and at least one barcode that identifies the antibody; b) hybridizing the oligonucleotide of the antibody-oligonucleotide conjugate to a recognition element, wherein the recognition element comprises a 5’ end and a 3’ end that are complementary to the at least one barcode of the oligonucleotide of the antibody-oligonucleotide conjugate, and a code that uniquely identifies the recognition element that is hybridized to the oligonucleotide of the antibody-oligonucleotide conjugate; c) ligating the 5’ end and the 3’ end of the recognition element that is hybridized to the oligonucleotide of the antibody-oligonucleotide conjugate, thereby generating a ligated recognition element; d) amplifying the ligated recognition element to generate a concatemeric amplification product; and e) determining a sequence of the code in the concatemeric amplification product, and using the sequence of the code to correlate the code with the presence of the protein of interest from the sample.

2. The method of claim 1, further comprising amplifying the oligonucleotide of the antibody- oligonucleotide conjugate bound to the protein of interest, thereby generating a barcoded amplicon.

3. The method of claim 2, further comprising hybridizing the barcoded amplicon to the recognition element.

4. The method of any one of claims 1-3, wherein a plurality of antibody-oligonucleotide conjugates are hybridized to a plurality of proteins of interest from the sample, and wherein a plurality of recognition elements are hybridized to the plurality of oligonucleotides of the plurality of antibody-oligonucleotide conjugates, for determining the presence of the plurality of proteins of interest from the sample.

5. The method of claim 4, further comprising amplifying the plurality of oligonucleotides of the plurality of antibody-oligonucleotide conjugates bound to the plurality of proteins of interest, thereby generating a plurality of barcoded amplicons.

6. The method of claim 5, further comprising hybridizing the plurality of barcoded amplicons to the plurality of recognition elements.

7. The method of any one of claims 1-6, wherein the sample comprises a blood sample, a tissue sample, a cell culture sample, a biopsy sample, or a buffy coat sample.

8. The method of any one of claims 1-6, wherein the sample comprises a lysate or a permeabilized sample.

9. The method of any one of claims 1-8, wherein the protein of interest is associated with one or more immune cells comprising one or more of a T lymphocyte, a B lymphocyte, or a natural killer lymphocyte, and wherein the one or more of the T lymphocyte, the B lymphocyte, or the natural killer lymphocyte is present in the sample.

10. The method of claim 9, wherein the one or more immune cells comprises the T lymphocyte.

11. The method of claim 10, wherein the T lymphocyte is selected from the group consisting of CD3, CD4, CD8, CD25, CTLA-4, IFNg, IL-10, IL-2, IL-6, LAG-3, PD-1, PD-L1, and TNFa.

12. The method of claim 4, wherein the plurality of the antibody-oligonucleotide conjugates are bound to proteins on an immune cell, wherein the proteins on the immune cell comprise two or more of CD3, CD4, CD8, CD25, CTLA-4, IFNg, IL-10, IL-2, IL-6, LAG-3, PD-1, PD-L1, or TNFa.

13. The method of claim 12, wherein the method is performed concurrently for two or more of CD3, CD4, CD8, CD25, CTLA-4, IFNg, IL-10, IL-2, IL-6, LAG-3, PD-1, PD-L1, or TNFa.

14. The method of claim 12, wherein the method is performed concurrently for IFNg, IL- 10, IL- 2, IL-6, LAG-3, PD-1, PD-L1, or TNFa.

15. The method of claim 12, wherein the method is performed concurrently for CD3, CD4, CD8, CD25, CTLA-4, LAG-3, PD-1, or PD-Ll.

16. The method of any one of claims 1-15, wherein the at least two functional sequences of the oligonucleotide are selected from the group consisting of a cleavage site sequence, an amplification primer binding site sequence, and a capture sequence.

17. The method of claim 1, wherein the at least two functional sequences of the oligonucleotide comprise amplification primer binding sites for generating barcoded amplicons.

18. The method of any one of claims 1-17, wherein the oligonucleotide comprises one barcode.

19. The method of any one of claims 1-17, wherein the oligonucleotide comprises two barcodes.

20. The method of any one of claims 1-19, wherein the at least two functional sequences of the oligonucleotide flank the at least one barcode.

21. The method of claim 2, wherein the amplifying the oligonucleotide of the antibody - oligonucleotide conjugate comprises polymerase chain reaction.

22. The method of any one of claims 1-21, wherein the amplifying the ligated recognition element comprises rolling circle amplification or multiple strand displacement amplification.

23. The method of any one of claims 1-22, wherein the code comprises one or more nucleic acid segments.

24. The method of claim 23, wherein the determining the sequence of the code comprises:(i) hybridizing one or more detection polynucleotides to the one or more nucleic acid segments, or portions thereof, of the code;(ii) imaging the hybridizing of the one or more detection polynucleotides to the one or more nucleic acid segments;(iii) generating one or more signals from the imaging; and(iv) decoding the one or more signals.

25. The method of claim 23, wherein the determining the sequence of the code comprises:(i) hybridizing a plurality of detection polynucleotides to the one or more nucleic acid segments, or portions thereof, of the code;(ii) imaging the of the plurality of detection polynucleotides to the one or more nucleic acid segments;(iii) generating a plurality of signals from the imaging; and(iv) decoding the plurality of signals.

26. The method of claim 24 or 25, wherein the decoding the one or more signals or the decoding the plurality of signals comprises soft decision decoding.

27. The method of claim 24 or 25, wherein the one or more detection polynucleotides or the plurality of detection polynucleotides comprise at least two different fluorescent moieties.

28. A composition, comprising: a) a recognition element, wherein the recognition element comprises: i) a 5’ end and a 3’ end adjacently hybridized to an oligonucleotide of an antibody - oligonucleotide conjugate, wherein the oligonucleotide comprises a barcode that identifies a protein that is recognized and bound by the antibody of the antibody-oligonucleotide conjugate; and ii) a code comprising one or more nucleic acid segments, wherein the code uniquely identifies the recognition element, wherein the 5’ end and the 3’ end are ligated together to generate a circularized, ligated recognition element.

29. The composition of claim 28, further comprising a ligase.

30. The composition of claim 28 or 29, further comprising an exonuclease.

31. A method for identifying a presence of a protein of interest in a sample, comprising: a) binding an antibody-oligonucleotide conjugate to the protein of interest in the sample, wherein the antibody-oligonucleotide conjugate comprises an antibody that recognizes and binds to the protein of interest and an oligonucleotide that is attached to the antibody, wherein theoligonucleotide comprises at least two functional sequences and one or more barcodes that identify the antibody; b) amplifying the oligonucleotide of the antibody-oligonucleotide conjugate, or a portion thereof, thereby generating a plurality of barcoded amplicons from the oligonucleotide of the antibody-oligonucleotide conjugate; c) hybridizing a barcoded amplicon of the plurality of barcoded amplicons to a recognition element, wherein the recognition element comprises a 5’ end and a 3’ end that are complementary to one or more barcodes of a barcoded amplicon of the plurality of barcoded amplicons, and a code that uniquely identifies the recognition element hybridized to the barcoded amplicon of the plurality of barcoded amplicons; d) ligating the 5’ end and 3’ end of the recognition element hybridized to the barcoded amplicon of the plurality of barcoded amplicons, thereby generating a ligated recognition element; e) amplifying the ligated recognition element to generate a concatemeric amplification product; and f) determining a sequence of the code in the concatemeric amplification product, and using the sequence of the code to correlate the code with the protein of interest in the sample.

32. The method of claim 31, wherein a plurality of antibody-oligonucleotide conjugates are hybridized to a plurality of proteins of interest from the sample, and wherein a plurality of recognition elements are hybridized to the plurality of barcoded amplicons, for determining the presence of a plurality of proteins of interest from the sample.

33. The method of claim 31 or 32, wherein the protein of interest is selected from the group consisting of CD3, CD4, CD8, CD25, CTLA-4, IFNg, IL-10, IL-2, IL-6, LAG-3, PD-1, PD-L1, and TNFa.

34. The method of claim 31 or 32, wherein the protein of interest comprises two or more of CD3, CD4, CD8, CD25, CTLA-4, IFNg, IL-10, IL-2, IL-6, LAG-3, PD-1, PD-L1, and TNFa.

35. The method of claim 31 or 32, wherein the method is performed concurrently for two or more of CD3, CD4, CD8, CD25, CTLA-4, IFNg, IL-10, IL-2, IL-6, LAG-3, PD-1, PD-L1, or TNFa.

36. The method of claim 31 or 32, wherein the method is performed concurrently for IFNg, IL- 10, IL-2, IL-6, LAG-3, PD-1, PD-L1, or TNFa.

37. The method of claim 31 or 32, wherein the method is performed concurrently for CD3, CD4, CD8, CD25, CTLA-4, LAG-3, PD-1, or PD-Ll.

38. The method of any one of claims 31-37, wherein the at least two functional sequences of the oligonucleotide of the antibody-oligonucleotide conjugate are selected from the group consisting of a cleavage site sequence, an amplification primer binding site sequence, and a capture sequence.

39. The method of claim 38, wherein the at least two functional sequences of the oligonucleotide comprise amplification primer binding sites for generating the plurality of barcoded amplicons.

40. The method of any one of claims 31-39, wherein the oligonucleotide comprises one barcode.

41. The method of any one of claims 31-39, wherein the oligonucleotide comprises two consecutive barcodes.

42. The method of any one of claims 31-41, wherein the at least two functional sequences of the oligonucleotide flank the one or more barcodes.

43. The method of claim 31, wherein the amplifying the oligonucleotide of the antibody- oligonucleotide conjugate comprises polymerase chain reaction.

44. The method of claim 31, wherein the amplifying the ligated recognition element comprises rolling circle amplification or multiple strand displacement amplification.

45. The method of any one of claims 31-44, wherein the code comprises one or more nucleic acid segments.

46. The method of claim 45, wherein the determining the sequence of the code comprises:(i) hybridizing one or more detection polynucleotides to the one or more nucleic acid segments, or portions thereof, of the code;(ii) imaging the hybridizing of the one or more detection polynucleotides to the one or more nucleic acid segments;(iii) generating one or more signals from the imaging the hybridizing; and(iv) decoding the one or more signals.

47. The method of claim 46, wherein the decoding the one or more signals comprises soft decision decoding.

48. The method of claim 31, wherein the determining the sequence of the code comprises next generation sequencing.

49. The method of claim 46, wherein the one or more detection polynucleotides comprises a fluorescent moiety.

50. The method of claim 49, wherein the fluorescent moiety is different for two or more of the one or more detection polynucleotides.

51. A method for identifying a presence of an immune system protein in a sample, comprising: a) binding an antibody-oligonucleotide conjugate to an immune system protein from the sample, wherein the antibody-oligonucleotide conjugate comprises an antibody that recognizes and binds to the immune system protein and an oligonucleotide that is attached to the antibody, wherein the oligonucleotide comprises one or more barcodes that identify the antibody; b) hybridizing the oligonucleotide of the antibody-oligonucleotide conjugate to a recognition element, wherein the recognition element comprises a 5’ end and a 3’ end that are complementary to the one or more barcodes of the oligonucleotide, and a code that uniquely identifies the recognition element that is hybridized to the oligonucleotide of the antibody- oligonucleotide conjugate; c) ligating the 5’ end and 3’ end of the recognition element hybridized to the oligonucleotide of the antibody-oligonucleotide conjugate, thereby generating a ligated recognition element; d) amplifying the ligated recognition element to generate a concatemeric amplification product; ande) determining a sequence of the code in the concatemeric amplification product, and using the sequence of the code to correlate the code with the presence of the immune system protein.

52. The method of claim 51, further comprising amplifying the oligonucleotide of the antibody- oligonucleotide conjugate, thereby generating a barcoded amplicon.

53. The method of claim 52, further comprising hybridizing the barcoded amplicon to the recognition element.

54. The method of any one of claims 51-53, wherein a plurality of antibody-oligonucleotides are hybridized to a plurality of immune system proteins from the sample, and wherein a plurality of recognition elements are hybridized to a plurality of oligonucleotides of the plurality of antibody-oligonucleotide conjugates, for determining the presence of the plurality of proteins from the sample.

55. The method of claim 54, further comprising amplifying the plurality of oligonucleotides of the plurality of antibody-oligonucleotide conjugates, thereby generating a plurality of barcoded amplicons.

56. The method of claim 55, further comprising hybridizing the plurality of barcoded amplicons to the plurality of recognition elements.

57. The method of claim 51, wherein the sample comprises a blood sample, a tissue sample, a cell culture sample, a biopsy sample, or a buffy coat sample.

58. The method of claim 51, wherein the sample comprises a lysate or a permeabilized sample.

59. The method of any one of claims 51-58, wherein the immune system protein is associated with one or more immune cells comprising one or more of a T lymphocyte, a B lymphocyte, or a natural killer lymphocyte.

60. The method of claim 59, wherein the one or more immune cells comprises the T lymphocyte.

61. The method of claim 60, wherein the T lymphocyte is selected from the group consisting of CD3, CD4, CD8, CD25, CTLA-4, IFNg, IL-10, IL-2, IL-6, LAG-3, PD-1, PD-L1, and TNFa.

62. The method of claim 61, wherein the T lymphocyte comprises two or more of CD3, CD4, CD8, CD25, CTLA-4, IFNg, IL-10, IL-2, IL-6, LAG-3, PD-1, PD-L1, or TNFa.

63. The method of claim 62, wherein the method is performed concurrently for two or more of CD3, CD4, CD8, CD25, CTLA-4, IFNg, IL-10, IL-2, IL-6, LAG-3, PD-1, PD-L1, or TNFa.

64. The method of claim 62, wherein the method is performed concurrently for IFNg, IL- 10, IL- 2, IL-6, LAG-3, PD-1, PD-L1, or TNFa.

65. The method of claim 62, wherein the method is performed concurrently for CD3, CD4, CD8, CD25, CTLA-4, LAG-3, PD-1, or PD-Ll.

66. The method of any one of claims 51-65, wherein the oligonucleotide of the antibody- oligonucleotide conjugate further comprises at least two functional sequences selected from the group consisting of a cleavage site sequence, an amplification primer binding site sequence, and a capture sequence.

67. The method of claim 66, wherein the at least two functional sequences comprise amplification primer binding sites for generating the barcoded amplicons.

68. The method of claim 51, wherein the oligonucleotide of the antibody-oligonucleotide conjugate comprises one barcode.

69. The method of claim 51, wherein the oligonucleotide of the antibody-oligonucleotide conjugate comprises two barcodes.

70. The method of any one of claims 66-69, wherein the at least two functional sequences of the oligonucleotide flank the one or more barcodes.

71. The method of claim 51, wherein amplifying the ligated recognition element comprises rolling circle amplification or multiple strand displacement amplification.

72. The method of any one of claim 51-71, wherein the code comprises one or more nucleic acid segments.

73. The method of claim 72, wherein the determining the sequence of the code comprises hybridizing one or more detection polynucleotides to the one or more nucleic acid segments of the code to produce one or more signals, and decoding the one or more signals.

74. The method of claim 73, wherein the decoding the one or more signals comprises soft decision decoding.

75. The method of claim 73, wherein the one or more detection polynucleotides comprise a fluorescent moiety.

76. The method of claim 75, further comprising imaging the fluorescent moiety of the hybridized detection polynucleotide, wherein the hybridizing of the one or more detection polynucleotides and the imaging is done for one or more cycles to generate a plurality of images.

77. The method of claim 76, wherein the plurality of images are combined and used as a recognition element profile.

78. The method of claim 77, further comprising soft decision decoding the recognition element profile.

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