Methods for high-plexity extracellular vesicle protein characterization through immunopcr / ngs

Abstract

Provided herein are methods for high-plexity EV surface protein characterization. Provided herein are also methods for determining the likelihood that a subject has a disease or condition, such as cancer.

Description

Attorney Docket No.: GH0256WOMETHODS FOR HIGH-PLEXTTY EXTRACELLULAR VESICLE PROTEIN CHARACTERIZATION THROUGH IMMUNOPCR / NGSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of US Provisional Patent Application No. 63 / 713,558, filed October 29, 2024, which is incorporated by reference herein in its entirety for all purposes.FIELD OF THE INVENTION

[0002] The present disclosure provides compositions and methods related to enriching extracellular vesicles in a sample. The present disclosure also provides compositions and methods for highly multi-plexed detection and quantification of surface proteins of extracellular vesicles (e.g., exosomes). In some embodiments, the proteins being assayed are from a subject having or suspected of having a disease or disorder, such as cancer.INTRODUCTION AND SUMMARY

[0003] Invasive diagnostic procedures, including biopsies, are commonly used for detecting or diagnosing cancer, ulcers, liver diseases, infections, transplant rejections, and other diseases and disorders in which analysis of cells or tissue from a possible site of a malady are analyzed for relevant features. Detection of diseases and disorders based on analysis of body fluids (“liquid biopsies”), such as blood, is an intriguing alternative. A liquid biopsy is noninvasive, sometimes requiring only a blood draw. However, it has been challenging to develop accurate and sensitive methods for analyzing proteins in liquid biopsy material in part because some of the same proteins released into body fluids due to disease are the same proteins that are normally present in body fluids.

[0004] Many types of diseases involve aberrant cell death or changes in cell death, including cancer, autoimmune diseases, infection and sepsis, myocardial infarction, ischemic injury, brain injury, liver disease, and neurodegenerative disease. Exosomes and other nanoscale vesicles secreted by cells, such as tumor cells, contain proteins and nucleic acids that can also provide important information about the state of the cell from which they originated. Identifying andAttorney Docket No.: GH0256WO quantifying molecules, such as proteins, from exosomes and other extracellular vesicles, can provide important information for detection of disease in a subject.

[0005] Current methods to characterize extracellular vesicles (EV) surface proteome are limited in targeting relatively low number of proteins, are low sample-throughput and / or associated with high costs, assay complexity and sample volume requirement.

[0006] The present disclosure aims to meet the need for improved analysis of molecules originating from dead or dying cells, such as from tumor cells. Improved detection of cancer markers in blood allows for more accurate detection of disorders (diagnosis) and therefore improved treatments. Methods disclosed herein can provide low-cost and high-throughput extracellular vesicle (EV) surface proteome characterization through immune-capture and immune-PCR or next generation sequencing (NGS). The number of surface proteins assayable can be order(s) of magnitude higher than current methods, and protein co-localization information important for performance in some diagnostic applications (e.g. cancer screening) is resolvable with approaches provided herein.

[0007] Accordingly, the following exemplary embodiments are provided.

[0008] Embodiment l is a method of analyzing extracellular vesicle (EV) markers in a sample, the method comprising: (a) obtaining an EV-containing sample; (b) enriching for EVs comprising a first EV marker using an immobilized first EV marker binding molecule that binds to the first EV marker, thereby obtaining immobilized EVs; (c) contacting the immobilized EVs with a set of second EV marker binding molecule-oligonucleotide conjugates to generate complexes of second EV marker binding molecules bound to the immobilized EVs; (d) removing unbound second EV marker binding molecule-oligonucleotide conjugates from the complexes of second EV marker binding molecules bound to the immobilized EVs; and (e) detecting the second EV marker binding molecules bound to the immobilized EVs.

[0009] Embodiment 2 is a method of analyzing EV markers in a sample, the method comprising: (a) obtaining an EV-containing sample; (b) enriching for EVs comprising a first EV marker using an immobilized first EV marker binding molecule that binds to the first EV marker, thereby obtaining immobilized EVs; (c) contacting the immobilized EVs with a set of second EV marker binding molecule-oligonucleotide conjugates to generate complexes of second EV marker binding molecules bound to the immobilized EVs; (d) releasing the immobilized EVs, thereby providing released EVs; (e) removing unbound second EV marker binding molecule-oligonucleotideAttorney Docket No.: GH0256WO conjugates from the complexes of second EV marker binding molecules bound to the released EVs using chromatographic purification; and (f) detecting the second EV marker binding molecules bound to the released EVs.

[0010] Embodiment 3 is the method of any one of the preceding embodiments, wherein the EV markers are surface protein markers.

[0011] Embodiment 4 is the method of any one of the preceding embodiments, wherein the set of second EV marker binding molecule-oligonucleotide conjugates comprises 2-100 EV marker binding molecule-oligonucleotide conjugates, optionally wherein the set of second EV marker binding molecule-oligonucleotide conjugates comprises 2-100, 10-50, 20-40, 25-35, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more than 60 EV marker binding molecule-oligonucleotide conjugates.

[0012] Embodiment 5 is the method of any one of the preceding embodiments, wherein one or more of the EV marker binding molecules comprise an antibody, a knottin, an aptamer, an affimer, an avimer, a nanobody, a DARPin, a monobody, or an affinity clamp.

[0013] Embodiment 6 is the method of any one of the preceding embodiments, wherein one or more of the EV marker binding molecules comprise an antibody.

[0014] Embodiment 7 is the method of any one of the preceding embodiments, wherein the EV marker binding molecule-oligonucleotide conjugates independently comprise an oligonucleotide comprising a first portion comprising a universal sequence region, a second portion comprising a unique sequence region that identifies an EV marker, and a third portion comprising a second universal sequence region.

[0015] Embodiment 8 is the method of the immediately preceding embodiment, wherein the second universal sequence region comprises a double stranded portion and a single stranded portion.

[0016] Embodiment 9 is the method of any one of embodiments 7-8, wherein the second universal sequence region comprises the 3’ end of the first oligonucleotide.

[0017] Embodiment 10 is the method of any one of embodiments 8-9, wherein the single stranded portion of the second universal sequence region is located 3’ of the double stranded portion of the second universal sequence region.Attorney Docket No.: GH0256WO

[0018] Embodiment 11 is the method of any one of embodiments 8-10, wherein the unique sequence region is distal to the single stranded portion of the second universal sequence region relative to the double stranded portion of the second universal sequence region.

[0019] Embodiment 12 is the method of any one of embodiments 7-11, wherein the unique sequence region comprises a molecular tag.

[0020] Embodiment 13 is the method of the immediately preceding embodiment, wherein the molecular tag is a molecular barcode.

[0021] Embodiment 14 is the method of any one of the preceding embodiments, wherein the immobilized first EV marker binding molecule is bound to a solid support.

[0022] Embodiment 15 is the method of the immediately preceding embodiment, wherein a capture nucleotide sequence is linked to the solid support.

[0023] Embodiment 16 is the method of the immediately preceding embodiment, wherein the capture nucleotide sequence can hybridize with a universal region of an EV marker binding molecule-oligonucleotide conjugate and thereby participate in proximity extension reactions.

[0024] Embodiment 17 is the method of the immediately preceding embodiment, wherein the proximity extension reaction yields an extension product, further wherein the extension product is amplified by PCR.

[0025] Embodiment 18 is the method any one of the preceding embodiments, wherein the detecting comprises high multiplex PCR, digital PCR, quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), or sequencing.

[0026] Embodiment 19 is the method of any one of the preceding embodiments, further comprising amplifying at least a portion of the oligonucleotides in the bound second EV marker binding molecule-oligonucleotide conjugates.

[0027] Embodiment 20 is the method of the immediately preceding embodiment, wherein the detecting comprises sequencing at least a portion of the amplified oligonucleotides.

[0028] Embodiment 21 is the method of the immediately preceding embodiment, wherein the sequencing comprises next generation sequencing (NGS).

[0029] Embodiment 22 is the method of any one of the preceding embodiments, wherein the sample is partitioned into a plurality of subsamples prior to step (b).

[0030] Embodiment 23 is the method of the immediately preceding embodiment, wherein steps (b)-(d) are performed in parallel for each subsample of the plurality of sub samples.Attorney Docket No.: GH0256WO

[0031] Embodiment 24 is the method of any one of the preceding embodiments, wherein (a) obtaining an EV-containing sample comprises obtaining a plurality of EV-containing samples, and wherein the method further comprises performing steps (b)-(d) in parallel for each sample of the plurality of samples.

[0032] Embodiment 25 is the method of the immediately preceding embodiment, wherein each EV-containing sample is obtained from a different tissue.

[0033] Embodiment 26 is the method of any one of embodiments 24-25, wherein the first EV marker is a tissue-specific marker and / or a cell type marker.

[0034] Embodiment 27 is the method of any one of embodiments 22-25, wherein a multiplexed sequencing is performed.

[0035] Embodiment 28 is the method of any one of embodiments 19-27, wherein a sample index is added in the amplification step.

[0036] Embodiment 29 is the method of any one of embodiments 19-28, wherein a sequencing adapter is added in the amplification step.

[0037] Embodiment 30 is the method of any one of embodiments 19-29, wherein a molecular tag specific to each EV marker binding molecule is added in the amplification step.

[0038] Embodiment 31 is the method of any one of the preceding embodiments, wherein the first EV marker is selected from CEA, Mucl, PD-L1, CD9, CD3, CD151, CD81, CD63, ALIX, CD40, Hsp60, TSG1, and phosphatidylserine.

[0039] Embodiment 32 is the method of any one of the preceding embodiments, wherein the second EV marker binding molecules bind to one or more of ALIX, CD40, TGS1, phosphatidylserine, Mucl, CD3, CD147, CD151, carcinoembryonic antigen (CEA), CD66a, CD326 (also known as epithelial cell adhesion molecule, EpCAM), CD49b, heat shock protein 60 (Hsp60), copine-3 (CPNE3), folate receptor alpha (FRa), CD24, vascular endothelial growth factor (VEGF), prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), epidermal growth factor receptor (EGFR), Glypican-3 (GPC-3), Glypican-1 (GPC-1), CD63, CD9, programmed death ligand 1 (PD-L1), leucine-rich alpha-2-glycoprotein 1 (LRG1), Epstein-Barr virus latent membrane protein 1 (LMP1), Epstein-Barr virus BamHI-A rightward frame 1 (BARF1), matrix metalloproteinase 9 (MMP9), ceruloplasmin (CP), Dickkopf-related protein 4 (DKK4), carbonic anhydrase 9 (CAIX), tumor-associated calcium signal transducer 2 (TACSTD2), CD36, CD44, Claudin 4, LI cell adhesion molecule (L1CAM), CD24, A disintegrinAttorney Docket No.: GH0256WO and metalloproteinase domain-containing protein 10 (ADAMI 0), extracellular matrix metalloproteinase inducer (EMMPRIN), transforming growth factor P-1 (TGFp-1), melanoma antigen 3 / 6 (MAGE-3 / 6), CD37, CD53, CD81, CD47, CD26, CD196, CD274, CD133 , and / or Ephrin A2.

[0040] Embodiment 33 is the method of any one of the preceding embodiments, wherein the second EV marker binding molecules bind to 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, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more,21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more,29 or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 or more,37 or more, 38 or more, 39 or more, 40 or more, 41 or more, 42 or more, 43 or more, 44 or more,45 or more, 46 or more, 47 or more, 48 or more, 49 or more, 50 or more, or each of ALIX, CD40, TGS1, phosphatidylserine, Mucl, CD3, CD147, CD151, carcinoembryonic antigen (CEA), CD66a, CD326 (also known as epithelial cell adhesion molecule, EpCAM), CD49b, heat shock protein 60 (Hsp60), copine-3 (CPNE3), folate receptor alpha (FRa), CD24, vascular endothelial growth factor (VEGF), prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), epidermal growth factor receptor (EGFR), Glypican-3 (GPC-3), Glypican-1 (GPC-1), CD63, CD9, programmed death ligand 1 (PD-L1), leucine-rich alpha-2-glycoprotein 1 (LRG1), Epstein-Barr virus latent membrane protein 1 (LMP1), Epstein-Barr virus BamHI-A rightward frame 1 (BARF1), matrix metalloproteinase 9 (MMP9), ceruloplasmin (CP), Dickkopf-related protein 4 (DKK4), carbonic anhydrase 9 (CAIX), tumor-associated calcium signal transducer 2 (TACSTD2), CD36, CD44, Claudin 4, LI cell adhesion molecule (L1CAM), CD24, A disintegrin and metalloproteinase domain-containing protein 10 (ADAM 10), extracellular matrix metalloproteinase inducer (EMMPRIN), transforming growth factor P-1 (TGFP-1), melanoma antigen 3 / 6 (MAGE-3 / 6), CD37, CD53, CD81, CD47, CD26, CD196, CD274, CD133, and / or Ephrin A2.

[0041] Embodiment 34 is the method of any one of the preceding embodiments, wherein the sample is a urine sample, an ascites sample, or a saliva sample.

[0042] Embodiment 35 is the method of any one of the preceding embodiments, wherein the sample is a blood sample.Attorney Docket No.: GH0256WO

[0043] Embodiment 36 is the method of the immediately preceding embodiment, wherein the blood sample is a whole blood sample, a plasma sample, a buffy coat sample, a leukapheresis sample, or a PBMC sample.

[0044] Embodiment 37 is the method of any one of the preceding embodiments, wherein the sample comprises plasma obtained from a blood sample.

[0045] Embodiment 38 is the method of any one of the preceding embodiments, wherein the sample comprises serum.

[0046] Embodiment 39 is the method of any one of the preceding embodiments, wherein the sample is a tissue sample.

[0047] Embodiment 40 is the method of the immediately preceding embodiment, wherein the tissue sample is a biopsy, a fine needle aspirate, or a formalin-fixed paraffin-embedded tissue sample.

[0048] Embodiment 41 is the method of any one of the preceding embodiments, wherein at least one of the EV markers is a cell type marker.

[0049] Embodiment 42 is the method of any one of the preceding embodiments, wherein the detecting step further comprises quantifying the level of one or more of the EV markers present in the sample.

[0050] Embodiment 43 is the method of any one of the preceding embodiments, wherein the sample is obtained from a subject.

[0051] Embodiment 44 is the method of the immediately preceding embodiment, wherein the subject is an animal.

[0052] Embodiment 45 is the method of any one of embodiments 43-44, wherein the subject is a human.

[0053] Embodiment 46 is the method of any one of the preceding embodiments, wherein at least one of the EV markers is associated with a disease or condition.

[0054] Embodiment 47 is the method of the immediately preceding embodiment, wherein the disease or condition is a cancer.

[0055] Embodiment 48 is the method of any one of embodiments 46-47, wherein the subject has or is at risk of having the disease or condition.Attorney Docket No.: GH0256WO

[0056] Embodiment 49 is the method of any one of embodiments 43-48, wherein the method comprises analyzing EV markers in a subsample of the sample or in a second sample obtained from the same subject from which the first sample is obtained.

[0057] Embodiment 50 is the method of any one of embodiments 43-49, comprising determining a likelihood that the subj ect has precancer.

[0058] Embodiment 51 is the method of any one of embodiments 43-50, comprising determining a likelihood that the subject has cancer.

[0059] Embodiment 52 is the method of any one of the preceding embodiments, wherein the sample is obtained from a subject who was previously diagnosed with a cancer and received one or more previous cancer treatments, optionally wherein the sample is obtained at one or more preselected time points following the one or more previous cancer treatments.

[0060] Embodiment 53 is the method of the immediately preceding embodiment, further comprising determining a cancer recurrence score, optionally wherein the cancer recurrence status of the subject is determined to be at risk for cancer recurrence when a cancer recurrence score is determined to be at or above a predetermined threshold or the cancer recurrence status of the subject is determined to be at lower risk for cancer recurrence when the cancer recurrence score is below the predetermined threshold.

[0061] Embodiment 54 is the method of the immediately preceding embodiment, further comprising comparing the cancer recurrence score of the subject with a predetermined cancer recurrence threshold, wherein the subject is classified as a candidate for a subsequent cancer treatment when the cancer recurrence score is above the cancer recurrence threshold or not a candidate for a subsequent cancer treatment when the cancer recurrence score is below the cancer recurrence threshold.BRIEF DESCRIPTION OF THE DRAWINGS

[0062] FIG. 1 is a schematic diagram of an example of a system suitable for use with some embodiments of the disclosure.

[0063] FIG. 2 shows the abundance of a plurality of EV markers assayed from human plasma samples as described in Example 2.Attorney Docket No.: GH0256WODETAILED DESCRIPTION OE CERTAIN EMBODIMENTS

[0064] Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with such embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the invention as defined by the appended claims.

[0065] Before describing the present teachings in detail, it is to be understood that the disclosure is not limited to specific compositions or process steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a nucleic acid” includes a plurality of nucleic acids, reference to “a cell” includes a plurality of cells, and the like.

[0066] Numeric ranges are inclusive of the numbers defining the range. Measured and measurable values are understood to be approximate, taking into account significant digits and the error associated with the measurement. Also, the use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the teachings.

[0067] Unless specifically noted in the above specification, embodiments in the specification that recite “comprising” various components are also contemplated as “consisting of’ or “consisting essentially of’ the recited components; embodiments in the specification that recite “consisting of’ various components are also contemplated as “comprising” or “consisting essentially of’ the recited components; and embodiments in the specification that recite “consisting essentially of’ various components are also contemplated as “consisting of’ or “comprising” the recited components (this interchangeability does not apply to the use of these terms in the claims).

[0068] The section headings used herein are for organizational purposes and are not to be construed as limiting the disclosed subject matter in any way. In the event that any document or other material incorporated by reference contradicts any explicit content of this specification, including definitions, this specification controls.Attorney Docket No.: GH0256WOT. Definitions

[0069] An “epitope” as used herein means the portion of a molecule or complex that is specifically bound by a binding molecule.

[0070] A “binding molecule” as used herein means a molecule capable of specifically binding an epitope. Binding molecules include, e.g., nanobodies, aptamers, affimers, DARPins, lectins and proteins comprising more than one polypeptide chain, such as antibodies.

[0071] “Cell type marker” as used herein means a molecule that is present in higher proportion in one or more cell types than in other cell types present in the same sample or than in any other cell type-10072] An “extracellular vesicle (EV)” as used herein refers to a membrane (e.g., lipid bilayer)- containing vesicle released (secreted) to the extracellular environment by different cell types. Extracellular vesicles (EV) encompass a number of different membraned vesicles produced by cells, the names of which include, for example, microvesicles, epididimosomes, argosomes, exosome-like vesicles, microparticles, promininosomes, prostasomes, dexosomes, texosomes, archeosomes, oncosomes, and exersomesectosomes, microparticles and shedding microvesicles. Extracellular vesicles (EV) circulate through body fluids, including blood, plasma, serum and urine. Circulating EV may contain exosomes and microvesicles (MV).

[0073] An “exosome” as used herein refers to a small membrane extracellular vesicle of ~30-300 nm or '40-120 nm diameter that is secreted from producing cells into the extracellular environment, as described initially by Trams, E. G. et al., 1981, Biochim. Biophys. Acta, 645(l):63-70. The surface (membrane surface) of an exosome comprises a lipid bilayer from the membrane of the donor cell, and the lumen of the exosome is topologically the same as the cytosol from the cell that produces the exosome. The exosome contains proteins, RNAs, lipids, and carbohydrates of the producing cell, though some may be modified or added to the exosome after its release from the cell, either through natural processes or by experimental manipulation. Illustrative exosome markers include Alix, TsglOl, tetraspanins (CD81, CD63, CD9), flotillin, synectin, or LAMP -2.

[0074] “Microvesicle” (abbreviated “MV”) as used herein refers to a single membrane vesicle secreted by different cell types. MV may have a diameter (or largest dimension where the particle is not spheroid) of between about 10 nm to about 5000 nm (e.g., between about 50 nm and 1500 nm, between about 75 nm and 1500 nm, between about 75 nm and 1250 nm, between about 50 nmAttorney Docket No.: GH0256WO and 1250 nm, between about 30 nm and 1000 nm, between about 50 nm and 1000 nm, between about 100 nm and 1000 nm, between about 50 nm and 750 nm, etc.). Microvesicles originate from cells, yet different subpopulations of microvesicles may exhibit different surface / lipid characteristics. Typically, at least part of the membrane of the microvesicle is directly obtained from a cell (also known as a donor cell). Microvesicles may originate from cells by membrane inversion, exocytosis, shedding, blebbing, and / or budding. Depending on the manner of generation (e.g., membrane inversion, exocytosis, shedding, or budding), microvesicles may exhibit different surface / lipid characteristics. Illustrative microvesicle markers include integrins, selectins and CD40. Microvesicles have been called by alternative names in the art, such as, for example, EV, exosomes, membrane particles, exosome-like particles, and apoptotic vesicles.

[0075] “Extracellular vesicle marker” or “EV marker” as used herein means a molecule, such as a protein, lipid, or carbohydrate, that is physically associated with or embedded in the outer membrane of an extracellular vesicle (e g. exosome) and is present in greater proportion in extracellular vesicle than on the outer membrane of intact live cells, cell debris, or in the soluble fraction of a sample.

[0076] “Extracellular vesicle marker binding molecule” or “EV marker binding molecule” as used herein means a molecule that specifically binds an EV marker. For example, an antibody that specifically binds an EV marker is an EV marker binding molecule. For example, an antibody that specifically binds an exosome marker is an exosome marker binding molecule. Binding molecules also include nanobodies, aptamers, affimers, DARPins, and the like.

[0077] Exosome marker” as used herein means a molecule, such as a protein, lipid, or carbohydrate, that is physically associated with or embedded in the outer membrane of an exosome and is present in greater proportion in exosomes than on the outer membrane of intact live cells, cell debris, or in the soluble fraction of a sample. Examples of exosome markers include but are not limited to tetraspanines, CD9, CD63, and CD8.

[0078] ‘ ‘Exosome marker binding molecule” as used herein means a molecule that specifically binds an exosome marker. For example, an antibody that specifically binds an exosome marker is an exosome marker binding molecule. Binding molecules also include nanobodies, aptamers, affimers, DARPins, and the like.

[0079] “Buffy coat” refers to the portion of a blood (such as whole blood) or bone marrow sample that contains all or most of the white blood cells and platelets of the sample. The buffy coat fraction1 !Attorney Docket No.: GH0256WO of a sample can be prepared from the sample using centrifugation, which separates sample components by density. For example, following centrifugation of a whole blood sample, the buffy coat fraction is situated between the plasma and erythrocyte (red blood cell) layers. The buffy coat can contain both mononuclear (e.g., T cells, B cells, NK cells, dendritic cells, and monocytes) and polymorphonuclear (e g., granulocytes such as neutrophils and eosinophils) white blood cells.

[0080] As used herein, “leukapheresis” refers to a procedure in which white blood cells (leukocytes) are isolated from a sample of blood collected from a subject. Leukapheresis may be performed, e.g., obtain cells for research, diagnostic, prognostic, or monitoring purposes, such as those described herein. Thus, as used herein, a “leukapheresis sample” refers to a sample comprising leukocytes collected from a subject using leukapheresis.

[0081] As used herein, “peripheral blood mononuclear cells” or “PBMCs” refers to immune cells having a single, round nucleus that originate in bone marrow and are found in the peripheral circulation. Such cells include, e.g., lymphocytes (T cells, B cells, and NK cells) as well as monocytes, and are isolated from blood samples (such as from a whole blood sample collected from a subject) using density gradient centrifugation.

[0082] “ Solid tissue” as used herein means tissue other than blood, blood components, other fluids such as lymph and interstitial fluid, and includes, e.g., epithelial tissue, connective tissue, muscle tissue, nervous tissue, and tissue of the colon, lung, breast, skin, prostate, stomach, pancreas, bladder, kidney, and liver. Solid tissue may be normal, precancerous, or cancerous (e.g., a malignant solid tumor such as a carcinoma or sarcoma).

[0083] “ Solid tissue cells” as used herein means cells in or derived from a solid tissue. Solid tissue cells exclude circulating cell types, such as cells normally present in blood or lymph. Examples of solid tissue cell types include but are not limited to colon, lung, breast, skin, prostate, stomach, pancreas, and liver cells.

[0084] “Cell-free DNA,” “cfDNA molecules,” or simply “cfDNA” include DNA molecules that naturally occur in a subject in extracellular form (e.g., in blood, serum, plasma, or other bodily fluids such as lymph, cerebrospinal fluid, urine, or sputum). While the cfDNA previously existed in a cell or cells in a large complex biological organism, e.g., a mammal, it has undergone release from the cell(s) into a fluid found in the organism, and may be obtained from a sample of the fluid without the need to perform an in vitro cell lysis step. cfDNA molecules may occur as DNA fragments.Attorney Docket No.: GH0256WO

[0085] As used herein, a “blood sample” refers to a sample comprising whole blood or a component thereof (e.g., plasma, serum, buffy coat, plasma pellet).

[0086] As used herein, “partitioning” of nucleic acids, such as DNA molecules, means separating, fractionating, sorting, or enriching a sample or population of nucleic acids into a plurality of subsamples or subpopulations of nucleic acids based on one or more modifications or features that is in different proportions in each of the plurality of subsamples or subpopulations. Partitioning may include physically partitioning nucleic acid molecules based on the presence or absence of one or more methylated nucleobases. A sample or population may be partitioned into one or more partitioned subsamples or subpopulations based on a characteristic that is indicative of a genetic or epigenetic change or a disease state.

[0087] As used herein, the form of the “originally isolated” sample refers to the composition or chemical structure of a sample at the time it was isolated and before undergoing any procedure that changes the chemical structure of the isolated sample. Similarly, a feature that is “originally present” in a molecule refers to a feature present in an “original molecule” or in molecules “originally comprising” the feature before the molecule undergoes any procedure that changes the chemical structure of the molecule.

[0088] As used herein, “base pairing specificity” refers to the standard DNA base (A, C, G, or T) for which a given base most preferentially pairs. For example, unmodified cytosine and 5- methylcytosine have the same base pairing specificity (i.e., specificity for G) whereas uracil and cytosine have different base pairing specificity because uracil has base pairing specificity for A while cytosine has base pairing specificity for G. The ability of uracil to form a wobble pair with G is irrelevant because uracil nonetheless most preferentially pairs with A among the four standard DNA bases.

[0089] As used herein, a “combination” comprising a plurality of members refers to either of a single composition comprising the members or a set of compositions in proximity, e.g., in separate containers or compartments within a larger container, such as a multiwell plate, tube rack, refrigerator, freezer, incubator, water bath, ice bucket, machine, or other form of storage.

[0090] “Capturing” one or more target molecules, such as one or more proteins or nucleic acids or one or more molecules comprising at least one target region refers to preferentially isolating or separating the one or more target molecules from non-target molecules.Attorney Docket No.: GH0256WO

[0091] As used herein, a “label” is a capture moiety, fluorophore, oligonucleotide, or other moiety that facilitates detection, separation, or isolation of that to which it is attached.

[0092] As used herein, a “capture moiety” is a molecule that allows affinity separation of molecules linked to the capture moiety from molecules lacking the capture moiety. Exemplary capture moieties include biotin, which allows affinity separation by binding to streptavidin linked or linkable to a solid phase or an oligonucleotide, which allows affinity separation through binding to a complementary oligonucleotide linked or linkable to a solid phase.

[0093] As used herein, a “tag” is a molecule or sequence containing information that indicates a feature of the molecule to which the tag is associated. For example, molecules can bear a sample tag (which distinguishes molecules in one sample from those in a different sample), a molecular tag / molecular barcode / barcode (which distinguishes different molecules from one another (in both unique and non-unique tagging scenarios), a partition tag (which distinguishes molecules in a partition from molecules in another partition), or a purification tag. As used herein, “nucleic acid tag” refers to a short nucleic acid (e.g., less than about 500 nucleotides, about 100 nucleotides, about 50 nucleotides, or about 10 nucleotides in length), used to distinguish nucleic acids from different samples (e.g., representing a sample index), distinguish nucleic acids from different partitions (e.g., representing a partition tag) or different nucleic acid molecules in the same sample (e.g., representing a molecular barcode), of different types, or which have undergone different processing. The nucleic acid tag comprises a predetermined, fixed, non-random, random or semirandom oligonucleotide sequence. Such nucleic acid tags may be used to label different nucleic acid molecules or different nucleic acid samples or sub-samples. Nucleic acid tags can be singlestranded, double-stranded, or at least partially double-stranded. Nucleic acid tags optionally have the same length or varied lengths. Nucleic acid tags can also include double-stranded molecules having one or more blunt-ends, include 5’ or 3’ single-stranded regions (e.g., an overhang), and / or include one or more other single-stranded regions at other locations within a given molecule. Nucleic acid tags can be attached to one end or to both ends of the other nucleic acids (e.g., sample nucleic acids to be amplified and / or sequenced). Nucleic acid tags can be decoded to reveal information such as the sample of origin, form, or processing of a given nucleic acid. For example, nucleic acid tags can also be used to enable pooling and / or parallel processing of multiple samples comprising nucleic acids bearing different molecular barcodes and / or sample indexes in which the nucleic acids are subsequently being deconvolved by detecting (e.g., reading) the nucleic acid tags.LiAttorney Docket No.: GH0256WONucleic acid tags can also be referred to as identifiers (e.g. molecular identifier, sample identifier). Additionally, or alternatively, nucleic acid tags can be used as molecular identifiers (e.g., to distinguish between different molecules or amplicons of different parent molecules in the same sample or sub-sample). This includes, for example, uniquely tagging different nucleic acid molecules in a given sample, or non-uniquely tagging such molecules. In the case of non-unique tagging applications, a limited number of tags (i.e., molecular barcodes) may be used to tag each nucleic acid molecule such that different molecules can be distinguished based on their endogenous sequence information (for example, start and / or stop positions where they map to a selected reference genome, a sub-sequence of one or both ends of a sequence, and / or length of a sequence) in combination with at least one molecular barcode. Typically, a sufficient number of different molecular barcodes are used such that there is a low probability (e.g., less than about a 10%, less than about a 5%, less than about a 1%, or less than about a 0.1% chance) that any two molecules may have the same endogenous sequence information (e.g., start and / or stop positions, subsequences of one or both ends of a sequence, and / or lengths) and also have the same molecular barcode. Terms such as “adapters having distinct molecular barcodes” encompass adapters for uniquely or non-uniquely tagging molecules, in that regardless of whether the adapters are for unique or non-unique tagging, distinct barcodes will be present in the population of adapters.

[0094] As used herein, a “target protein” is a protein whose presence or absence is detected.

[0095] “Specifically binds” in the context of binding molecule (e.g., a protein, primer, probe, or other oligonucleotide), and a target protein or sequence means that under appropriate binding conditions, the binding molecule binds to its target to form a stable complex, while at the same time formation of stable non-target complexes is minimized. For example, a primer or probe hybridizes to a target sequence or replicate thereof to a sufficiently greater extent than to a nontarget sequence, to ultimately enable capture or detection of the target sequence. Appropriate binding conditions are well-known in the art, may be predicted based on sequence composition, or can be determined by using routine testing methods (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) at §§ 1.90-1.91, 7.37-7.57, 9.47-9.51 and 11.47-11.57, particularly §§ 9.50-9.51, 11.12- 11.13, 11.45-11.47 and 11.55-11.57, incorporated by reference herein).

[0096] “Substantially free” means free to a sufficient extent that the relevant properties are not meaningfully impacted by the presence of a minor impurity.Attorney Docket No.: GH0256WO

[0097] “Immunoassay” as used herein means an assay or method comprising contacting a molecule or sample with an antibody in order to test the function or detect, identify, and / or quantify the presence of one or more components of the sample. Examples of immunoassays may include but are not limited to enzyme-linked immunosorbent assays (ELISAs), sandwich assays, eletrochemiluminescence (ECL) assays, and multiplex assays.

[0098] An “antibody” as used herein is used broadly encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigenbinding activity.

[0099] An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab’-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.

[0100] A protein or nucleic acid is “produced by a tumor” if it originated from a tumor cell. DNA that originated from a tumor cell is “circulating tumor DNA” (“ctDNA”). Tumor cells are neoplastic cells that originated from a tumor, regardless of whether they remain in the tumor or become separated from the tumor (as in the cases, e.g., of metastatic cancer cells and circulating tumor cells).

[0101] A “target region” in the context of a nucleic acid refers to a genomic locus targeted for identification and / or capture, for example, by using probes (e.g., through sequence complementarity). A “target region set” or “set of target regions” refers to a plurality of genomic loci targeted for identification and / or capture, for example, by using a set of probes (e.g., through sequence complementarity).

[0102] “Sequence-variable target regions” refer to target regions that may exhibit changes in sequence such as nucleotide substitutions (i.e., single nucleotide variations), insertions, deletions, or gene fusions or transpositions in neoplastic cells (e.g., tumor cells and cancer cells) relative to normal cells. A sequence-variable target region set is a set of sequence-variable target regions. In some embodiments, the sequence-variable target regions are target regions that may exhibit changes that affect less than or equal to 50 contiguous nucleotides, e.g., less than or equal to 40, 30, 20, 10, 5, 4, 3, 2, or 1 nucleotides.Attorney Docket No.: GH0256WO

[0103] “Epigenetic target regions” refers to target regions that may show sequence-independent differences in different cell or tissue types (e.g., different types of immune cells) or in neoplastic cells (e.g., tumor cells and cancer cells) relative to normal cells; or that may show sequenceindependent differences in DNA, such as cfDNA, from different cell types or from subjects having cancer relative to DNA, such as cfDNA, from healthy subjects, or in cfDNA originating from different cell or tissue types that ordinarily do not substantially contribute to cfDNA (e.g., immune, lung, colon, etc.) relative to background cfDNA (e.g., cfDNA that originated from hematopoietic cells). Examples of sequence-independent changes include, but are not limited to, changes in methylation (increases or decreases), nucleosome distribution, cfDNA fragmentation patterns, CCCTC-binding factor (“CTCF”) binding, transcription start sites, and regulatory protein binding regions. Epigenetic target region sets thus include, but are not limited to, hypermethylation variable target region sets, hypomethylation variable target region sets, and fragmentation variable target region sets, such as CTCF binding sites and transcription start sites. For present purposes, loci susceptible to neoplasia-, tumor-, or cancer-associated focal amplifications and / or gene fusions may also be included in an epigenetic target region set because detection of a change in copy number by sequencing or a fused sequence that maps to more than one locus in a reference genome tends to be more similar to detection of exemplary epigenetic changes discussed above than detection of nucleotide substitutions, insertions, or deletions, e.g., in that the focal amplifications and / or gene fusions can be detected at a relatively shallow depth of sequencing because their detection does not depend on the accuracy of base calls at one or a few individual positions. An epigenetic target region set is a set of epigenetic target regions.

[0104] The “capture yield” of a collection of probes for a given target set refers to the amount (e.g., amount relative to another target set or an absolute amount) of nucleic acid corresponding to the target set that the collection of probes captures under typical conditions. Exemplary typical capture conditions are an incubation of the sample nucleic acid and probes at 65°C for 10-18 hours in a small reaction volume (about 20 uL) containing stringent hybridization buffer. The capture yield may be expressed in absolute terms or, for a plurality of collections of probes, relative terms. When capture yields for a plurality of sets of target regions are compared, they are normalized for the footprint size of the target region set (e.g., on a per-kilobase basis). Thus, for example, if the footprint sizes of first and second target regions are 50 kb and 500 kb, respectively (giving a normalization factor of 0.1), then the DNA corresponding to the first target region set is capturedAttorney Docket No.: GH0256WO with a higher yield than DNA corresponding to the second target region set when the mass per volume concentration of the captured DNA corresponding to the first target region set is more than 0.1 times the mass per volume concentration of the captured DNA corresponding to the second target region set. As a further example, using the same footprint sizes, if the captured DNA corresponding to the first target region set has a mass per volume concentration of 0.2 times the mass per volume concentration of the captured DNA corresponding to the second target region set, then the DNA corresponding to the first target region set was captured with a two-fold greater capture yield than the DNA corresponding to the second target region set.

[0105] The term “methylation” or “DNA methylation” refers to addition of a methyl group to a nucleobase in a nucleic acid molecule. In some embodiments, methylation refers to addition of a methyl group to a cytosine at a CpG site (cytosine-phosphate-guanine site (i.e., a cytosine followed by a guanine in a 5’ -> 3’ direction of the nucleic acid sequence). In some embodiments, DNA methylation refers to addition of a methyl group to adenine, such as in N6-methyladenine. In some embodiments, DNA methylation is 5-methylation (modification of the 5th carbon of the 6-carbon ring of cytosine). In some embodiments, 5-methylation refers to addition of a methyl group to the 5C position of the cytosine to create 5-methylcytosine (5mC). In some embodiments, methylation comprises a derivative of 5mC. Derivatives of 5mC include, but are not limited to, 5- hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), and 5-caryboxylcytosine (5-caC). In some embodiments, DNA methylation is 3C methylation (modification of the 3rd carbon of the 6- carbon ring of cytosine). In some embodiments, 3C methylation comprises addition of a methyl group to the 3C position of the cytosine to generate 3-methylcytosine (3mC). Methylation can also occur at non CpG sites, for example, methylation can occur at a CpA, CpT, or CpC site. DNA methylation can change the activity of methylated DNA region. For example, when DNA in a promoter region is methylated, transcription of the gene may be repressed. DNA methylation is critical for normal development and abnormality in methylation may disrupt epigenetic regulation. The disruption, e.g., repression, in epigenetic regulation may cause diseases, such as cancer. Promoter methylation in DNA may be indicative of cancer.

[0106] The term “hypermethylation” refers to an increased level or degree of methylation of nucleic acid molecule(s) relative to the other nucleic acid molecules within a population (e.g., sample) of nucleic acid molecules. In some embodiments, hypermethylated DNA can include DNAAttorney Docket No.: GH0256WO molecules comprising at least 1 methylated residue, at least 2 methylated residues, at least 3 methylated residues, at least 5 methylated residues, or at least 10 methylated residues.

[0107] The term “hypomethylation” refers to a decreased level or degree of methylation of nucleic acid molecule(s) relative to the other nucleic acid molecules within a population (e.g., sample) of nucleic acid molecules. In some embodiments, hypomethylated DNA includes unmethylated DNA molecules. In some embodiments, hypomethylated DNA can include DNA molecules comprising 0 methylated residues, at most 1 methylated residue, at most 2 methylated residues, at most 3 methylated residues, at most 4 methylated residues, or at most 5 methylated residues.

[0108] The terms “agent that recognizes a modified nucleobase in DNA,” such as an “agent that recognizes a modified cytosine in DNA” refers to a molecule or reagent that binds to or detects one or more modified nucleobases in DNA, such as methyl cytosine. A “modified nucleobase” is a nucleobase that comprises a difference in chemical structure from an unmodified nucleobase. In the case of DNA, an unmodified nucleobase is adenine, cytosine, guanine, or thymine. In some embodiments, a modified nucleobase is a modified cytosine. In some embodiments, a modified nucleobase is a methylated nucleobase. In some embodiments, a modified cytosine is a methyl cytosine, e.g., a 5-methyl cytosine. In such embodiments, the cytosine modification is a methyl. Agents that recognize a methyl cytosine in DNA include but are not limited to “methyl binding reagents,” which refer herein to reagents that bind to a methyl cytosine. Methyl binding reagents include but are not limited to methyl binding domains (MBDs) and methyl binding proteins (MBPs) and antibodies specific for methyl cytosine. In some embodiments, such antibodies bind to 5-methyl cytosine in DNA. In some such embodiments, the DNA may be single-stranded or double-stranded.

[0109] As used herein, “amplify,” “amplifying,” or “amplification” refers to a process by which extra or multiple copies of a particular polynucleotide are formed. Amplification methods can include any suitable methods known in the art. As used herein, a nucleic acid molecule amplified using “methylation-preserving amplification” substantially maintains its methylation status postamplification.

[0110] As used herein, a “primer-extended product,” when referring to primers that anneal to at least one target region, means a nucleic acid strand formed by extension of a primer annealed to a DNA or RNA target region. In some embodiments, a primer-extended product is a significant primer-extended product or is formed by significant primer extension, meaning that the resultingAttorney Docket No.: GH0256WO nucleic acid strand has sufficient additional length (e.g., at least 10, 15, 20, 30, 40, 50, 60, 75, or 100 nucleotides in addition to the length of the original primer) to be detected and / or identified using methods described herein.[OHl] As used herein, “adjacent” nucleosides or oligonucleotides are nucleosides or oligonucleotides that are next to each other, with no intervening nucleosides. For example, “adjacent” nucleosides may be covalently linked together within a nucleic acid or oligonucleotide, or they may be unlinked but are next to each other because they are annealed to or hybridized to adjacent linked nucleosides of a nucleic acid. “Adjacent” oligonucleotides may likewise be linked together or unlinked to each other but annealed to or hybridized to adjacent, linked portions of a nucleic acid.

[0112] As used herein, “mutation” refers to a variation from a known reference sequence and includes mutations such as, for example, single nucleotide variants (SNVs), and insertions or deletions (indels). A mutation can be a germline or somatic mutation. In some embodiments, a reference sequence for purposes of comparison is a wildtype genomic sequence of the species of the subject providing a test sample, typically the human genome.

[0113] As used herein, the terms “neoplasm” and “tumor” are used interchangeably. They refer to abnormal growth of cells in a subject. A neoplasm or tumor can be benign, potentially malignant, or malignant. A malignant tumor is referred to as a cancer or a cancerous tumor.

[0114] As used herein, “next-generation sequencing” or “NGS” refers to sequencing technologies having increased throughput as compared to traditional Sanger- and capillary electrophoresisbased approaches, for example, with the ability to generate hundreds of thousands of relatively small sequence reads at a time. Some examples of next-generation sequencing techniques include, but are not limited to, sequencing by synthesis, sequencing by ligation, and sequencing by hybridization. In some embodiments, next-generation sequencing includes the use of instruments capable of sequencing single molecules. Examples of commercially available instruments for performing next-generation sequencing include, but are not limited to, NextSeq, HiSeq, NovaSeq, MiSeq, Ion PGM and Ion GeneStudio S5.

[0115] As used herein, DNA that is “not immobilized” or that is “free in solution” refers to DNA that is not bound covalently or non-covalently to a solid support, such as a bead. Such DNA may be free in solution during any step (such as all steps) of the disclosed methods.Attorney Docket No.: GH0256WO

[0116] As used herein, “polynucleotide”, “nucleic acid”, “nucleic acid molecule”, or “oligonucleotide” refers to a linear polymer of nucleosides (including deoxyribonucleosides, ribonucleosides, or analogs thereof) joined by inter-nucleosidic linkages. Typically, a polynucleotide comprises at least three nucleosides. Oligonucleotides often range in size from a few monomeric units, e.g., 3-4, to hundreds of monomeric units. Whenever a polynucleotide is represented by a sequence of letters, such as “ATGCCTG”, the nucleotides are in 5’ to 3’ order from left to right, and in the case of DNA, “A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotes deoxythymidine, unless otherwise noted. The letters A, C, G, and T may be used to refer to the bases themselves, to nucleosides, or to nucleotides comprising the bases.

[0117] As used herein, “processing” refers to a set of steps used to generate a library of nucleic acids that is suitable for sequencing. The set of steps can include, but are not limited to, partitioning, end repairing, addition of sequencing adapters, tagging, and / or PCR amplification of nucleic acids.

[0118] As used herein, a “level,” “quantity,” or a “quantitative measure” refers to an absolute or relative measure. A quantitative measure can be, without limitation, a number, a statistical measurement (e.g., frequency, mean, median, standard deviation, or quantile), or a degree or a relative quantity (e.g., high, medium, and low). A quantitative measure can be a ratio of two quantitative measures. A quantitative measure can be a linear combination of quantitative measures. A quantitative measure may be a normalized measure.

[0119] As used herein, “reference sequence” refers to a known sequence used for purposes of comparison with experimentally determined sequences. For example, a known sequence can be an entire genome, a chromosome, or any segment thereof. A reference sequence can align with a single contiguous sequence of a genome or chromosome or chromosome arm or can include noncontiguous segments that align with different regions of a genome or chromosome.

[0120] As used herein, “sample” means anything capable of being analyzed by the methods and / or systems disclosed herein.

[0121] As used herein, “sequencing” refers to any of a number of technologies used to determine the sequence (e.g., the identity and order of monomer units) of a biomolecule, e.g., a nucleic acid such as DNA or RNA. Examples of sequencing methods include, but are not limited to, targeted sequencing, single molecule real-time sequencing, exon or exome sequencing, intron sequencing,Attorney Docket No.: GH0256WO electron microscopy-based sequencing, panel sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, whole-genome sequencing, sequencing by hybridization, pyrosequencing, duplex sequencing, cycle sequencing, single-base extension sequencing, solid-phase sequencing, high-throughput sequencing, massively parallel signature sequencing, emulsion PCR, co-amplification at lower denaturation temperature- PCR (COLD-PCR), multiplex PCR, sequencing by reversible dye terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, short-read sequencing, single-molecule sequencing, sequencing-by-synthesis, real-time sequencing, reverseterminator sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLiD™ sequencing, MS-PET sequencing, and a combination thereof. In some embodiments, sequencing can be performed by a gene analyzer such as, for example, gene analyzers commercially available from Illumina, Inc., Pacific Biosciences, Inc., or Applied Biosystems / Thermo Fisher Scientific, among many others.

[0122] As used herein, “sequence information” in the context of a nucleic acid polymer means the order and identity of monomer units (e.g., nucleotides, etc.) in that polymer.

[0123] As used herein, the terms “somatic mutation” or “somatic variation” are used interchangeably. They refer to a mutation in the genome that occurs after conception. Somatic mutations can occur in any cell of the body except germ cells and accordingly, are not passed on to progeny.

[0124] As used herein, “subject” refers to an animal, such as a mammalian species (e.g., human) or avian (e.g., bird) species, or other organism, such as a plant. More specifically, a subject can be a vertebrate, e.g., a mammal such as a mouse, a primate, a simian or a human. Animals include farm animals (e.g., production cattle, dairy cattle, poultry, horses, pigs, and the like), sport animals, and companion animals (e.g., pets or support animals). A subject can be a healthy individual, an individual that has or is suspected of having a disease or a predisposition to the disease, or an individual in need of therapy or suspected of needing therapy. The terms “individual” or “patient” are intended to be interchangeable with “subject”. For example, a subject can be an individual who has been diagnosed with having a cancer, is going to receive a cancer therapy, and / or has received at least one cancer therapy. The subject can be in remission of a cancer. As another example, the subject can be an individual who is diagnosed of having an autoimmune disease.Attorney Docket No.: GH0256WO

[0125] As used herein, an “asymmetric adapter” is a double stranded adapter in which the two strands are not completely complementary or are otherwise distinguishable such that synthesis of a complementary sequence of one strand of the adapter results in a sequence that is distinguishable from the sequence of the other strand of the adapter. Examples of asymmetric adapters are Y- shaped adapters and bubble adapters.

[0126] As used herein, a “Y-shaped adapter” refers to an adapter comprising two DNA strands comprising complementary and non-complementary parts, wherein the non-complementary parts form single-stranded arms. The adapter can be attached to a sample or insert DNA molecule, e.g., by ligation, such that the complementary (double-stranded) part of the adapter is proximal to the sample or insert DNA molecule. Prior to attachment, the double stranded portion of the Y-shaped adapter may have a blunt end or an overhang, e.g., of one to three nucleotides. The single stranded arms may or may not be of identical length.

[0127] As used herein, a “bubble adapter” refers to an adapter comprising two DNA strands comprising a non-complementary part flanked by complementary parts, such that the adapter has a single stranded region located between double-stranded regions. The adapter can be attached to a sample or insert DNA molecule, e.g., by ligation, such that one of the complementary (doublestranded) parts of the adapter is proximal to the sample or insert DNA molecule. Prior to attachment, the double stranded portion of the Y-shaped adapter that would be attached to the insert or sample molecule may have a blunt end or an overhang, e.g., of one to three nucleotides. The single stranded portions of the two strands may or may not be of identical length.

[0128] A “type of dNTP” refers to a dNTP comprising a specific base, including A, T, G or C. Accordingly, wherein an end repair reaction is performed with dNTPs, wherein at least one type of dNTP comprises a modified base, the end repair reaction may be performed using dCTP comprising 5mC, and dATP, dTTP and dGTP all comprising non-modified bases.

[0129] Bases of the “same identity” refer to the same base, regardless of modification status of that base. For example, cytosine is considered to be the “same identity” as 5-methylcytosine (5mC) and / or 5-hydroxymethyl-cytosine (5hmC), despite them having different modification statuses.

[0130] The terms “or a combination thereof’ and “or combinations thereof’ as used herein refers to any and all permutations and combinations of the listed terms preceding the term. For example, “A, B, C, or combinations thereof’ is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC,Attorney Docket No.: GH0256WO or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CAB ABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0131] “ Or” is used in the inclusive sense, i.e., equivalent to “and / or,” unless the context requires otherwise.II. EXEMPLARY METHODSA. Overview

[0132] The present disclosure provides compositions and methods related to enriching / isolating EVs containing known surface marker of interest and then probed for presence of additional surface markers of interest with a known set of antibody-oligonucleotide conjugates (Ab-oligos). In some embodiments, unbound Ab-oligos are washed away and retained bound antibody- oligonucleotide conjugates are subjected to nucleic acid manipulation and detection assay, such as NGS, which can resolve and quantify many different surface protein targets (likely 100s or more, potentially up to millions). Certain methods disclosed herein enable a low cost, high sensitivity and high throughput 'proteome-wide' EV surface characterization. Certain methods disclosed herein can be used as a diagnostic tool (due to low cost, high sensitivity and high throughput) and / or biomarker discovery tool because of high targeting capability for cancer screening and other cancer / non-cancer diagnostic applications.

[0133] In some embodiments, methods disclosed herein comprise steps of : (a) obtaining an EV- containing sample; (b) enriching for EVs comprising a first EV marker using an immobilized first EV marker binding molecule that binds to the first EV marker, thereby obtaining immobilized EVs; (c) contacting the immobilized EVs with a set of second EV marker binding molecule- oligonucleotide conjugates to generate complexes of second EV marker binding molecules bound to the immobilized EVs; (d) removing unbound second EV marker binding molecule- oligonucleotide conjugates from the complexes of second EV marker binding molecules bound to the immobilized EVs; and (e) detecting the second EV marker binding molecules bound to the immobilized EVs. In some embodiments, the EV markers are surface protein markers.

[0134] In some embodiments, the set of second EV marker binding molecule-oligonucleotide conjugates comprises 2-100 EV marker binding molecule-oligonucleotide conjugates, optionallyAttorney Docket No.: GH0256WO wherein the set of second EV marker binding molecule-oligonucleotide conjugates comprises 2- 100, 10-50, 20-40, 25-35, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more than 60 EV marker binding molecule-oligonucleotide conjugates. In some embodiments, one or more of the EV marker binding molecules comprise an antibody, a knottin, an aptamer, an affimer, an avimer, a nanobody, a DARPin, a monobody, or an affinity clamp. In some embodiments, one or more of the EV marker binding molecules comprises an antibody.

[0135] In some embodiments, the EV marker binding molecule-oligonucleotide conjugates independently comprise an oligonucleotide comprising a first portion comprising a universal sequence region, a second portion comprising a unique sequence region that identifies an EV marker, and a third portion comprising a second universal sequence region. In some embodiments, the second universal sequence region comprises a double stranded portion and a single stranded portion. In some embodiments, the second universal sequence region comprises the 3’ end of the first oligonucleotide. In some embodiments, the single stranded portion of the second universal sequence region is located 3’ of the double stranded portion of the second universal sequence region. In some embodiments, the unique sequence region is distal to the single stranded portion of the second universal sequence region relative to the double stranded portion of the second universal sequence region. In some embodiments, the unique sequence region comprises a molecular tag. In some embodiments, the molecular tag is a molecular barcode.

[0136] In some embodiments, the detecting comprises high multiplex PCR, digital PCR, quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), or sequencing. In some embodiments, the methods provided herein further comprise amplifying at least a portion of the oligonucleotides in the bound second EV marker binding molecule-oligonucleotide conjugates. In some embodiments, the detecting comprises sequencing at least a portion of the amplified oligonucleotides. In some embodiments, the sequencing comprises next generation sequencing (NGS).

[0137] In some embodiments, the sample is partitioned into a plurality of subsamples prior to step (b). In some embodiments, steps (b)-(d) are performed in parallel for each subsample of the plurality of subsamples. In some embodiments, (a) obtaining an EV-containing sample comprises obtaining a plurality of EV-containing samples, and wherein the method further comprises performing steps (b)-(d) in parallel for each sample of the plurality of samples. In someAttorney Docket No.: GH0256WO embodiments, each EV-containing sample is obtained from a different tissue. Tn some embodiments, the first EV marker is a tissue-specific marker and / or a cell type marker.

[0138] In some embodiments, a multiplexed sequencing is performed. In some embodiments, sample index is added in the amplification step. In some embodiments, a sequencing adapter is added in the amplification step. In some embodiments, a molecular tag specific to each EV marker binding molecule is added in the amplification step.

[0139] In some embodiments, the first EV marker is selected from CEA, Mucl, PD-L1, CD9, CD3, CD151, CD81, CD63, ALIX, CD40, Hsp60, TSG1, and phosphatidylserine.

[0140] In some embodiments, the second EV marker binding molecules bind to one or more of ALIX, CD40, TGS1, phosphatidylserine, Mucl, CD3, CD147, CD151, carcinoembryonic antigen (CEA), CD66a, CD326 (also known as epithelial cell adhesion molecule, EpCAM), CD49b, heat shock protein 60 (Hsp60), copine-3 (CPNE3), folate receptor alpha (FRa), CD24, vascular endothelial growth factor (VEGF), prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), epidermal growth factor receptor (EGFR), Glypican-3 (GPC-3), Glypican-1 (GPC-1), CD63, CD9, programmed death ligand 1 (PD-L1), leucine-rich alpha-2-glycoprotein 1 (LRG1), Epstein-Barr virus latent membrane protein 1 (LMP1), Epstein-Barr virus BamHI-A rightward frame 1 (BARF1), matrix metalloproteinase 9 (MMP9), ceruloplasmin (CP), Dickkopf- related protein 4 (DKK4), carbonic anhydrase 9 (CAIX), tumor-associated calcium signal transducer 2 (TACSTD2), CD36, CD44, Claudin 4, LI cell adhesion molecule (L1CAM), CD24, A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), extracellular matrix metalloproteinase inducer (EMMPRIN), transforming growth factor P-1 (TGF -1), melanoma antigen 3 / 6 (MAGE-3 / 6), CD37, CD53, CD81, CD47, CD26, CD 196, CD274, CD 133, and / or Ephrin A2.

[0141] In some embodiments, the second EV marker binding molecules bind to 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, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 or more, 37 or more, 38 or more, 39 or more, 40 or more, 41 or more, 42 or more, 43 or more, 44 or more, 45 or more, 46 or more, 47 or more, 48 or more, 49 or more, 50 or more, or each of ALIX, CD40, TGS1, phosphatidyl serine, Mucl, CD3, CD147, CD151,Attorney Docket No.: GH0256WO carcinoembryonic antigen (CEA), CD66a, CD326 (also known as epithelial cell adhesion molecule, EpCAM), CD49b, heat shock protein 60 (Hsp60), copine-3 (CPNE3), folate receptor alpha (FRa), CD24, vascular endothelial growth factor (VEGF), prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), epidermal growth factor receptor (EGFR), Glypican- 3 (GPC-3), Glypican-1 (GPC-1), CD63, CD9, programmed death ligand 1 (PD-L1), leucine-rich alpha-2-glycoprotein 1 (LRG1), Epstein-Barr virus latent membrane protein 1 (LMP1), Epstein- Barr virus BamHI-A rightward frame 1 (BARF1), matrix metalloproteinase 9 (MMP9), ceruloplasmin (CP), Dickkopf-related protein 4 (DKK4), carbonic anhydrase 9 (CAIX), tumor- associated calcium signal transducer 2 (TACSTD2), CD36, CD44, Claudin 4, LI cell adhesion molecule (LI CAM), CD24, A disintegrin and metalloproteinase domain-containing protein 10 (ADAM 10), extracellular matrix metalloproteinase inducer (EMMPRIN), transforming growth factor p-1 (TGFP-1), melanoma antigen 3 / 6 (MAGE-3 / 6), CD37, CD53, CD81, CD47, CD26, CD196, CD274, CD133, and / or Ephrin A2.

[0142] In some embodiments, the sample is a urine sample, an ascites sample, or a saliva sample. In some embodiments, the sample is a blood sample. In some embodiments, the blood sample is a whole blood sample, a plasma sample, a buffy coat sample, a leukapheresis sample, or a PBMC sample. In some embodiments, the sample comprises plasma obtained from a blood sample. In some embodiments, the sample comprises serum. In some embodiments, the sample is a tissue sample. In some embodiments, the tissue sample is a biopsy, a fine needle aspirate, or a formalin- fixed paraffin-embedded tissue sample.

[0143] In some embodiments, at least one of the EV markers is a cell type marker.In some embodiments, the detecting step further comprises quantifying the level of one or more of the EV markers present in the sample.

[0144] In some embodiments, the sample is obtained from a subject. In some embodiments, the subject is an animal. In some embodiments, the subject is a human. In some embodiments, at least one of the EV markers is associated with a disease or condition. In some embodiments, the disease or condition is a cancer. In some embodiments, the subject has or is at risk of having the disease or condition. In some embodiments, the method comprises analyzing EV markers in a subsample of the sample or in a second sample obtained from the same subject from which the first sample is obtained. In some embodiments, the method comprises determining a likelihood that the subject has precancer. In some embodiments, the method comprises determining a likelihood that theAttorney Docket No.: GH0256WO subject has cancer. Tn some embodiments, the sample is obtained from a subject who was previously diagnosed with a cancer and received one or more previous cancer treatments, optionally wherein the sample is obtained at one or more preselected time points following the one or more previous cancer treatments. In some embodiments, the method comprises determining a cancer recurrence score, optionally wherein the cancer recurrence status of the subject is determined to be at risk for cancer recurrence when a cancer recurrence score is determined to be at or above a predetermined threshold or the cancer recurrence status of the subject is determined to be at lower risk for cancer recurrence when the cancer recurrence score is below the predetermined threshold. In some embodiments, the method comprises comparing the cancer recurrence score of the subject with a predetermined cancer recurrence threshold, wherein the subject is classified as a candidate for a subsequent cancer treatment when the cancer recurrence score is above the cancer recurrence threshold or not a candidate for a subsequent cancer treatment when the cancer recurrence score is below the cancer recurrence threshold.Additional embodiments:

[0145] In some embodiments, multiplexed sequencing is performed. In such embodiments, the sample index is added in the amplification step. In some embodiments, the enriching step can be multiplexed - a sample may be run in parallel through steps (b) to (d) and a DNA tag specific to each type of antibody that the EVs are enriched for can be added during amplification step.

[0146] In some embodiments, direct immunostaining of a sample or exosome-enriched sample is performed; followed by chromatographic purification of the complexes of second EV marker binding molecules bound to immobilized EVs to remove excess reagent. In some embodiments, the methods disclosed herein improve signal by reducing potential background signal arising from residual antibody reagent that may be incompletely washed away. In some embodiments, provided herein is a method of analyzing EV markers in a sample, the method comprising: (a) obtaining an EV-containing sample; (b) enriching for EVs comprising a first EV marker using an immobilized first EV marker binding molecule that binds to the first EV marker, thereby obtaining immobilized EVs; (c) contacting the immobilized EVs with a set of second EV marker binding molecule- oligonucleotide conjugates to generate complexes of second EV marker binding molecules bound to the immobilized EVs; (d) releasing the immobilized EVs, thereby providing released EVs; (e) removing unbound second EV marker binding molecule-oligonucleotide conjugates from the complexes of second EV marker binding molecules bound to the released EVs usingAttorney Docket No.: GH0256WO chromatographic purification; and (f) detecting the second EV marker binding molecules bound to the released EVs.

[0147] In some embodiments, the immobilized support may comprise a capture nucleotide sequence, in addition to the first EV marker binding molecule. In some embodiments, the immobilized capture nucleotide sequence may further participate in proximity extension reactions with exosome-bound antibody-oligonucleotide complexes following immunocapture. In some embodiments, the capture nucleotide sequence can hybridize with a universal region of an EV marker binding molecule-oligonucleotide conjugate and thereby participate in proximity extension reactions. In some embodiments, the proximity extension reaction yields an extension product. The extension product may be further amplified by PCR to introduce sample indexes and sequencing adapters. Such embodiments may provide a means to reduce non-specific background signal. In some embodiments, the immobilized first EV marker binding molecule is bound to a solid support. In some embodiments, a capture nucleotide sequence is linked to the solid support. In some embodiments, the capture nucleotide sequence can hybridize with a universal region of an EV marker binding molecule-oligonucleotide conjugate and thereby participate in proximity extension reactions. In some embodiments, the proximity extension reaction yields an extension product, further wherein the extension product is amplified by PCR.

[0148] In some embodiments, the methods disclosed herein are used in multicancer detection, by detecting multiple exosome species originating from a variety of tissue sources. A sample may be partitioned into multiple immunocapture reactions, which individually are designed to capture exosomes originating from different tissues e.g. by using tissue-specific protein markers. The separate exosome capture reactions are contacted with multiple oligonucleotide barcoded antibodies directed against other exosome-specific and tissue-specific protein targets, followed by sample-specific indexing PCR and introduction of sequencing adapters.

[0149] Methods of the present disclosure can be implemented using, or with the aid of, computer systems. FIG. 1 shows a computer system 101 that is programmed or otherwise configured to implement the methods of the present disclosure.

[0150] FIG. 2 shows the abundance of plurality of EV markers assayed from human plasma samples using the methods provided herein.Attorney Docket No.: GH0256WOB. Subjects

[0151] The disclosure relates to methods of analyzing EV markers in a sample obtained from a subject. In some embodiments, the sample is obtained from a subject having a cancer or a precancer, an infection, transplant rejection, or other disease directly or indirectly affecting the immune system. In some embodiments, the sample is obtained from a subject suspected of having a cancer or a precancer, an infection, transplant rejection, or other disease directly or indirectly affecting the immune system. In some embodiments, the sample is obtained from a subject having a tumor. In some embodiments, the sample is obtained from a subject suspected of having a tumor. In some embodiments, the sample is obtained from a subject having neoplasia. In some embodiments, the sample is obtained from a subject suspected of having neoplasia. In some embodiments, the sample is obtained from a subject in remission from a tumor, cancer, or neoplasia (e.g., following chemotherapy, surgical resection, radiation, or a combination thereof). In any of the foregoing embodiments, the cancer, tumor, or neoplasia or suspected cancer, tumor, or neoplasia may be of the lung, colon, rectum, kidney, breast, prostate, or liver. In some embodiments, the cancer, tumor, or neoplasia or suspected cancer, tumor, or neoplasia is of the lung. In some embodiments, the cancer, tumor, or neoplasia or suspected cancer, tumor, or neoplasia is of the colon or rectum. In some embodiments, the cancer, tumor, or neoplasia or suspected cancer, tumor, or neoplasia is of the breast. In some embodiments, the cancer, tumor, or neoplasia or suspected cancer, tumor, or neoplasia is of the prostate. In some embodiments, the sample is obtained from a subject having a stage I cancer, stage II cancer, stage III cancer or stage IV cancer.

[0152] In any of the foregoing embodiments, the subject may be a human subject. The subject may be a human, a mammal, an animal, a primate, rodent (including mice and rats), or other common laboratory, domestic, companion, service or agricultural animal, for example a rabbit, dog, cat, horse, cow, sheep, goat or pig.

[0153] In some embodiments, the subject may have an infection, a transplant rejection, or other disease or disorder related to changes in the immune system. The subject may not have cancer or a detectable cancer symptom. The subject may have been treated with one or more cancer therapy, e.g., any one or more of chemotherapies, antibodies, vaccines or biologies. The subject may be in remission. The subject may or may not be diagnosed as being susceptible to cancer or any cancer- associated genetic mutations / disorders.Attorney Docket No.: GH0256WOC. Analysis

[0154] The present methods can be used to diagnose the presence of conditions, particularly cancer or precancer, in a subject, to characterize conditions (e.g., staging cancer or determining heterogeneity of a cancer), monitor response to treatment of a condition, effect prognosis risk of developing a condition or subsequent course of a condition. The present disclosure can also be useful in determining the efficacy of a particular treatment option. Successful treatment options may increase the amount of copy number variation, rare mutations, or target proteins detected in a subject’s blood if the treatment is successful as more cancers may die and shed DNA and proteins, among other things. In other examples, this may not occur. In another example, perhaps certain treatment options may be correlated with profiles of protein post-translational modifications and / or genetic profiles of cancers over time. This correlation may be useful in selecting a therapy.

[0155] Additionally, if a cancer is observed to be in remission after treatment, the present methods can be used to monitor residual disease or recurrence of disease.

[0156] The types and number of cancers that may be detected may include blood cancers, brain cancers, lung cancers, skin cancers, nose cancers, throat cancers, liver cancers, bone cancers, lymphomas, pancreatic cancers, skin cancers, bowel cancers, rectal cancers, thyroid cancers, bladder cancers, kidney cancers, mouth cancers, stomach cancers, solid state tumors, heterogeneous tumors, homogenous tumors and the like. Type and / or stage of cancer can be detected from genetic variations including mutations, rare mutations, indels, copy number variations, transversions, translocations, recombination, inversion, deletions, aneuploidy, partial aneuploidy, polyploidy, chromosomal instability, chromosomal structure alterations, gene fusions, chromosome fusions, gene truncations, gene amplification, gene duplications, chromosomal lesions, DNA lesions, abnormal changes in nucleic acid chemical modifications, abnormal changes in epigenetic patterns, and abnormal changes in nucleic acid 5-methylcytosine.

[0157] The disclosed methods can be combined with analysis of one or more additional biomarkers. In some embodiments, the disclosed methods are combined with one or more methods, such as but not limited to, methods for assessing DNA methylation patterns, DNA mutations (such as somatic mutations), nucleic acid fragmentation patterns, non-coding RNA (such as micro RNAs (miRNAs), ribosomal RNAs, transfer RNAs, small nucleolar RNAs (snow RNAs), and / or small nuclear RNAs (snRNAs)) levels, and / or cell type proportions / levels, cellular locations, and / or structural modifications of one or more proteins (such as in a sample from aAttorney Docket No.: GH0256WO subject). In some embodiments, the disclosed methods are combined with one or more analyses of genetic variations including mutations, rare mutations, indels, rearrangements, copy number variations, transversions, translocations, recombinations, inversion, deletions, aneuploidy, partial aneuploidy, polyploidy, chromosomal instability, chromosomal structure alterations, gene fusions, chromosome fusions, gene truncations, gene amplification, gene duplications, chromosomal lesions, DNA lesions, abnormal changes in nucleic acid chemical modifications, abnormal changes in epigenetic patterns, and / or abnormal changes in nucleic acid 5-methylcytosine.

[0158] In some embodiments, a method described herein comprises identifying the presence of target proteins and / or DNA produced by a tumor (or neoplastic cells, or cancer cells) or by precancer cells.

[0159] Genetic data can also be used for characterizing a specific form of cancer. Cancers are often heterogeneous in both composition and staging. Genetic profile data may allow characterization of specific sub-types of cancer that may be important in the diagnosis or treatment of that specific sub-type. This information may also provide a subject or practitioner clues regarding the prognosis of a specific type of cancer and allow either a subject or practitioner to adapt treatment options in accord with the progress of the disease. Some cancers can progress to become more aggressive and genetically unstable. Other cancers may remain benign, inactive or dormant. The system and methods of this disclosure may be useful in determining disease progression. This information may also provide a subject or practitioner clues regarding the prognosis of a specific type of cancer and allow either a subject or practitioner to adapt treatment options in accord with the progress of the disease. Some cancers progress, becoming more aggressive and genetically unstable. Other cancers may remain benign, inactive or dormant. The system and methods of this disclosure may be useful in determining disease progression.

[0160] The present methods are useful in determining the efficacy of a particular treatment option. The present methods can also be used for detecting, e.g., variations in EV-associated target molecules in conditions other than cancer. Further, the methods of the disclosure may be used to characterize the heterogeneity of an abnormal condition in a subject, the method comprising generating a profile of co-localized EV-associated target molecules in the subject. In some cases, including but not limited to cancer, a disease may be heterogeneous. Disease cells may not be identical. In the example of cancer, some tumors are known to comprise different types of tumor cells, some cells in different stages of the cancer.Attorney Docket No.: GH0256WO

[0161] Further, the methods of the disclosure may be used to characterize the heterogeneity of an abnormal condition in a subject. Such methods can include, e.g., generating a profile of target proteins derived from the subject, wherein the profile comprises a plurality of data resulting from detections described herein, optionally in combination with additional data, e.g., epigenetic changes, copy number variation, and / or mutations. In some embodiments, an abnormal condition is cancer or precancer. In some embodiments, the abnormal condition may be one resulting in a heterogeneous genomic population. In the example of cancer, some tumors are known to comprise tumor cells in different stages of the cancer. In other examples, heterogeneity may comprise multiple foci of disease. Again, in the example of cancer, there may be multiple tumor foci, perhaps where one or more foci are the result of metastases that have spread from a primary site.

[0162] The present methods can be used to generate a profile, fingerprint or set of data that is a summation of information derived from different cells in a heterogeneous disease. This set of data may comprise target protein post-translational modifications, identities, levels, copy number variation, epigenetic variation, or other mutation analyses alone or in combination.

[0163] The present methods can be used to diagnose, prognose, monitor or observe cancers, or other diseases. In some embodiments, the methods herein do not involve the diagnosing, prognosing or monitoring a fetus and as such are not directed to non-invasive prenatal testing. In other embodiments, these methodologies may be employed in a pregnant subject to diagnose, prognose, monitor or observe cancers or other diseases in an unborn subject whose DNA and other polynucleotides may co-circulate with maternal molecules.D. Analysis of DNA; Partitioning the sample into a plurality of subsamples

[0164] In some embodiments described herein, the disclosed methods further comprise analyzing DNA in a sample (which may be a separate sample from the same subject or the same sample). For example, analyzing DNA such as cell-free DNA in combination with analyzing post- translationally modified proteins may improve the specificity and / or sensitivity of methods that detect abnormal states, such as the presence of a disease. In such methods, different forms of DNA (e.g., hypermethylated and hypomethylated DNA) can be physically partitioned based on one or more characteristics of the DNA. This approach can be used to determine, for example, whether certain sequences are hypermethylated or hypomethylated. Detecting aberrant features in DNA (whether sequence-based, epigenetic, or both) while also detecting aberrant levels of one or more post-translationally modified proteins may provide greater specificity and / or sensitivity forAttorney Docket No.: GH0256WO identifying an abnormal state than detecting the DNA features alone or levels of one or more post- translationally modified proteins alone.

[0165] Methylation profiling can involve determining methylation patterns across different regions of the genome. For example, after partitioning molecules based on extent of methylation (e.g., relative number of methylated nucleobases per molecule) and sequencing, the sequences of molecules in the different partitions can be mapped to a reference genome. This can show regions of the genome that, compared with other regions, are more highly methylated or are less highly methylated. In this way, genomic regions, in contrast to individual molecules, may differ in their extent of methylation.

[0166] Partitioning nucleic acid molecules in a sample can increase a rare signal, e.g., by enriching rare nucleic acid molecules that are more prevalent in one partition of the sample. For example, a genetic variation present in hypermethylated DNA but less (or not) present in hypomethylated DNA can be more easily detected by partitioning a sample into hypermethylated and hypomethylated nucleic acid molecules. By analyzing multiple partitions of a sample, a multidimensional analysis of a single molecule can be performed and hence, greater sensitivity can be achieved. Partitioning may include physically partitioning nucleic acid molecules into partitions or subsamples based on the presence or absence of one or more methylated nucleobases. A sample may be partitioned into partitions or subsamples based on a characteristic that is indicative of differential gene expression or a disease state. A sample may be partitioned based on a characteristic, or combination thereof that provides a difference in signal between a normal and diseased state during analysis of nucleic acids, e.g., cell free DNA (cfDNA), non-cfDNA, tumor DNA, circulating tumor DNA (ctDNA) and cell free nucleic acids (cfNA).

[0167] In some embodiments, hypermethylation and / or hypomethylation variable epigenetic target regions are analyzed to determine whether they show differential methylation characteristic of tumor cells or cells of a type that does not normally contribute to the DNA sample being analyzed (such as cfDNA), and / or particular immune cell types.

[0168] In some instances, heterogeneous DNA in a sample is partitioned into two or more partitions (e.g., at least 3, 4, 5, 6 or 7 partitions). In some embodiments, each partition is differentially tagged. Tagged partitions can then be pooled together for collective sample prep and / or sequencing. The partitioning-tagging-pooling steps can occur more than once, with each round of partitioning occurring based on a different characteristics (examples provided herein),Attorney Docket No.: GH0256WO and tagged using differential tags that are distinguished from other partitions and partitioning means. In other instances, the differentially tagged partitions are separately sequenced.

[0169] In some embodiments, sequence reads from differentially tagged and pooled DNA are obtained and analyzed in silico. Tags are used to sort reads from different partitions. Analysis to detect genetic variants can be performed on a partition-by-partition level, as well as whole nucleic acid population level. For example, analysis can include in silico analysis to determine genetic variants, such as CNV, SNV, indel, fusion in nucleic acids in each partition. In some instances, in silico analysis can include determining chromatin structure. For example, coverage of sequence reads can be used to determine nucleosome positioning in chromatin. Higher coverage can correlate with higher nucleosome occupancy in genomic region while lower coverage can correlate with lower nucleosome occupancy or nucleosome depleted region (NDR).

[0170] Examples of characteristics that can be used for partitioning include sequence length, methylation level, nucleosome binding, sequence mismatch, immunoprecipitation, and / or proteins that bind to DNA. Resulting partitions can include one or more of the following nucleic acid forms: single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), shorter DNA fragments and longer DNA fragments. In some embodiments, partitioning based on a cytosine modification (e.g., cytosine methylation) or methylation generally is performed and is optionally combined with at least one additional partitioning step, which may be based on any of the foregoing characteristics or forms of DNA. In some embodiments, a heterogeneous population of nucleic acids is partitioned into nucleic acids with one or more epigenetic modifications and without the one or more epigenetic modifications. Examples of epigenetic modifications include presence or absence of methylation; level of methylation; type of methylation (e.g., 5-methylcytosine versus other types of methylation, such as adenine methylation and / or cytosine hydroxymethylation); and association and level of association with one or more proteins, such as histones. Alternatively or additionally, a heterogeneous population of nucleic acids can be partitioned into nucleic acid molecules associated with nucleosomes and nucleic acid molecules devoid of nucleosomes. Alternatively or additionally, a heterogeneous population of nucleic acids may be partitioned into single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). Alternatively, or additionally, a heterogeneous population of nucleic acids may be partitioned based on nucleic acid length (e.g., molecules of up to 160 bp and molecules having a length of greater than 160 bp).Attorney Docket No.: GH0256WO

[0171] The agents used to partition populations of nucleic acids within a sample can be affinity agents, such as antibodies with the desired specificity, natural binding partners or variants thereof (Bock et al., Nat Biotech 28: 1106-1114 (2010); Song et al., Nat Biotech 29: 68-72 (2011)), or artificial peptides selected e.g., by phage display to have specificity to a given target. In some embodiments, the agent used in the partitioning is an agent that recognizes a modified nucleobase. In some embodiments, the modified nucleobase recognized by the agent is a modified cytosine, such as a methylcytosine (e.g., 5-methylcytosine). In some embodiments, the modified nucleobase recognized by the agent is a product of a procedure that affects the first nucleobase in the DNA differently from the second nucleobase in the DNA of the sample. In some embodiments, the modified nucleobase may be a “converted nucleobase,” meaning that its base pairing specificity was changed by a procedure. For example, certain procedures convert unmethylated or unmodified cytosine to dihydrouracil, or more generally, at least one modified or unmodified form of cytosine undergoes deamination, resulting in uracil (considered a modified nucleobase in the context of DNA) or a further modified form of uracil. Examples of partitioning agents include antibodies, such as antibodies that recognize a modified nucleobase, which may be a modified cytosine, such as a methylcytosine (e.g., 5-methylcytosine). In some embodiments, the partitioning agent is an antibody that recognizes a modified cytosine other than 5-methylcytosine, such as 5- carboxylcytosine (5caC). Alternative partitioning agents include methyl binding domain (MBDs) and methyl binding proteins (MBPs) as described herein, including proteins such as MeCP2.

[0172] Additional, non-limiting examples of partitioning agents are histone binding proteins which can separate nucleic acids bound to histones from free or unbound nucleic acids. Examples of histone binding proteins that can be used in the methods disclosed herein include RBBP4, RbAp48 and SANT domain peptides.

[0173] In some embodiments, partitioning can comprise both binary partitioning and partitioning based on degree / level of modifications. For example, methylated fragments can be partitioned by methylated DNA immunoprecipitation (MeDIP), or all methylated fragments can be partitioned from unmethylated fragments using methyl binding domain proteins (e.g., MethylMiner Methylated DNA Enrichment Kit (ThermoFisher Scientific). Subsequently, additional partitioning may involve eluting fragments having different levels of methylation by adjusting the salt concentration in a solution with the methyl binding domain and bound fragments. As salt concentration increases, fragments having greater methylation levels are eluted.Attorney Docket No.: GH0256WO

[0174] In some instances, the final partitions are enriched in nucleic acids having different extents of modifications (overrepresentative or underrepresentative of modifications). Overrepresentation and underrepresentation can be defined by the number of modifications bom by a nucleic acid relative to the median number of modifications per strand in a population. For example, if the median number of 5-methylcytosine residues in nucleic acid in a sample is 2, a nucleic acid including more than two 5-methylcytosine residues is overrepresented in this modification and a nucleic acid with 1 or zero 5-methylcytosine residues is underrepresented. The effect of the affinity separation is to enrich for nucleic acids overrepresented in a modification in a bound phase and for nucleic acids underrepresented in a modification in an unbound phase (i.e. in solution). The nucleic acids in the bound phase can be eluted before subsequent processing.

[0175] When using MeDIP or MethylMiner®Methylated DNA Enrichment Kit (ThermoFisher Scientific) various levels of methylation can be partitioned using sequential elutions. For example, a hypomethylated partition (no methylation) can be separated from a methylated partition by contacting the nucleic acid population with the MBD from the kit, which is attached to magnetic beads. The beads are used to separate out the methylated nucleic acids from the non- methylated nucleic acids. Subsequently, one or more elution steps are performed sequentially to elute nucleic acids having different levels of methylation. For example, a first set of methylated nucleic acids can be eluted at a salt concentration of 160 mM or higher, e.g., at least 150 mM, at least 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1000 mM, or 2000 mM. After such methylated nucleic acids are eluted, magnetic separation is once again used to separate higher level of methylated nucleic acids from those with lower level of methylation. The elution and magnetic separation steps can be repeated to create various partitions such as a hypomethylated partition (enriched in nucleic acids comprising no methylation), a methylated partition (enriched in nucleic acids comprising low levels of methylation), and a hyper methylated partition (enriched in nucleic acids comprising high levels of methylation).

[0176] In some methods, nucleic acids bound to an agent used for affinity separation based partitioning are subjected to a wash step. The wash step washes off nucleic acids weakly bound to the affinity agent. Such nucleic acids can be enriched in nucleic acids having the modification to an extent close to the mean or median (i.e., intermediate between nucleic acids remaining bound to the solid phase and nucleic acids not binding to the solid phase on initial contacting of the sample with the agent).Attorney Docket No.: GH0256WO

[0177] The affinity separation results in at least two, and sometimes three or more partitions of nucleic acids with different extents of a modification. While the partitions are still separate, the nucleic acids of at least one partition, and usually two or three (or more) partitions are linked to nucleic acid tags, usually provided as components of adapters, with the nucleic acids in different partitions receiving different tags that distinguish members of one partition from another. The tags linked to nucleic acid molecules of the same partition can be the same or different from one another. But if different from one another, the tags may have part of their code in common so as to identify the molecules to which they are attached as being of a particular partition.

[0178] For further details regarding portioning nucleic acid samples based on characteristics such as methylation, see WO2018 / 119452, which is incorporated herein by reference.

[0179] In some embodiments, the partitioning is performed by contacting the nucleic acids with a methyl binding domain (“MBD”) of a methyl binding protein (“MBP”). In some such embodiments, the nucleic acids are contacted with an entire MBP. In some embodiments, an MBD binds to 5-methylcytosine (5mC), and an MBP comprises an MBD and is referred to interchangeably herein as a methyl binding protein or a methyl binding domain protein. In some embodiments, MBD is coupled to paramagnetic beads, such as Dynabeads® M-280 Streptavidin via a biotin linker. Partitioning into fractions with different extents of methylation can be performed by eluting fractions by increasing the NaCl concentration.

[0180] In some embodiments, bound DNA is eluted by contacting the antibody or MBD with a protease, such as proteinase K. This may be performed instead of or in addition to elution steps using NaCl as discussed above.

[0181] Examples of agents that recognize a modified nucleobase contemplated herein include, but are not limited to:

[0182] (a) MeCP2 is a protein that preferentially binds to 5-methyl-cytosine over unmodified cytosine.

[0183] (b) RPL26, PRP8 and the DNA mismatch repair protein MHS6 preferentially bind to 5- hydroxymethyl -cytosine over unmodified cytosine.

[0184] (c) FOXK1, FOXK2, FOXP1, FOXP4 and FOXI3 preferably bind to 5 -formyl -cytosine over unmodified cytosine (lurlaro et al., Genome Biol. 14: R119 (2013)).

[0185] (d) Antibodies specific to one or more methylated or modified nucleobases or conversion products thereof, such as 5mC, 5caC, or DHU.Attorney Docket No.: GH0256WO

[0186] Tn general, elution is a function of the number of modifications, such as the number of methylated sites per molecule, with molecules having more methylation eluting under increased salt concentrations. To elute the DNA into distinct populations based on the extent of methylation, one can use a series of elution buffers of increasing NaCl concentration. Salt concentration can range from about 100 nm to about 2500 mMNaCl. In one embodiment, the process results in three (3) partitions. Molecules are contacted with a solution at a first salt concentration and comprising a molecule comprising an agent that recognizes a modified nucleobase, which molecule can be attached to a capture moiety, such as streptavidin. At the first salt concentration a population of molecules will bind to the agent and a population will remain unbound. The unbound population can be separated as a “hypomethylated” population. For example, a first partition enriched in hypomethylated form of DNA is that which remains unbound at a low salt concentration, e.g., 100 mM or 160 mM. A second partition enriched in intermediate methylated DNA is eluted using an intermediate salt concentration, e.g., between 100 mM and 2000 mM concentration. This is also separated from the sample. A third partition enriched in hypermethylated form of DNA is eluted using a high salt concentration, e.g., at least about 2000 mM.

[0187] In some embodiments, a monoclonal antibody raised against 5-methylcytidine (5mC) is used to purify methylated DNA. DNA is denatured, e.g., at 95°C in order to yield single- stranded DNA fragments. Protein G coupled to standard or magnetic beads as well as washes following incubation with the anti-5mC antibody are used to immunoprecipitate DNA bound to the antibody. Such DNA may then be eluted. Partitions may comprise unprecipitated DNA and one or more partitions eluted from the beads.

[0188] In some embodiments, the partitions of DNA are desalted and concentrated in preparation for enzymatic steps of library preparation.E. Adapter ligation or addition; tagging

[0189] In some embodiments, the disclosed methods further comprise analyzing DNA in a sample (which may be a separate sample from the same subject or the same sample). In such methods, adapters may be added to the DNA. In some embodiments, the adapters enable next generation sequencing (NGS). This may be done concurrently with an amplification procedure, e.g., by providing the adapters in a 5’ portion of a primer (where PCR is used, this can be referred to as library prep-PCR or LP-PCR). In some embodiments, adapters are added by other approaches, such as ligation. In some such methods, prior to partitioning or prior to capturing, first adaptersAttorney Docket No.: GH0256WO are added to the nucleic acids by ligation to the 3’ ends thereof, which may include ligation to single-stranded DNA. The adapter can be used as a priming site for second-strand synthesis, e.g., using a universal primer and a DNA polymerase. A second adapter can then be ligated to at least the 3’ end of the second strand of the now double-stranded molecule. In some embodiments, the first adapter comprises an affinity tag, such as biotin, and nucleic acid ligated to the first adapter is bound to a solid support (e.g., bead), which may comprise a binding partner for the affinity tag such as streptavidin. For further discussion of a related procedure, see Gansauge et al., Nature Protocols 8:737-748 (2013). Commercial kits for sequencing library preparation compatible with single-stranded nucleic acids are available, e.g., the Accel-NGS® Methyl-Seq DNA Library Kit from Swift Biosciences. In some embodiments, after adapter ligation, nucleic acids are amplified.

[0190] Preferably, the adapters include different tags of sufficient numbers that the number of combinations of tags results in a low probability e.g., 95, 99 or 99.9% of two nucleic acids with the same start and stop points receiving the same combination of tags. Adapters, whether bearing the same or different tags, can include the same or different primer binding sites, but preferably adapters include the same primer binding site.

[0191] In some embodiments, the single-stranded DNA library preparation is performed in a one- step combined phosphorylation / ligation reaction, e.g., as described in Troll et al., BMC Genomics, 20: 1023 (2019), available at https: / / doi.org / 10.1186 / sl2864-019-6355-0. This method, called Single Reaction Single-stranded LibrarY (“SRSLY,”) can be performed without end-polishing. SRSLY may be useful for converting short and fragmented DNA molecules, e.g., cfDNA fragments, into sequencing libraries while retaining native lengths and ends. The SRSLY method can create sequencing libraries (e.g., Illumina sequencing libraries) from fragmented or degraded template (input) DNA. In particular embodiments, template DNA is first heat denatured and then immediately cold shocked to render the template DNA molecules single-stranded. The DNA can be maintained as single- stranded throughout the ligation reaction by the inclusion of a thermostable single-stranded binding protein (SSB). Next, the template DNA, which at this point can be singlestranded and coated with SSB, is placed in a phosphorylation / ligation dual reaction with directional dsDNA NGS adapters that contain single-stranded overhangs. Both the forward and reverse sequencing adapters can share similar structures but differ in which termini is unblocked in order to facilitate proper ligations. Both sequencing adapters can comprise a dsDNA portion and a single-stranded splint overhang of random nucleotides that occurs on the 3-prime terminus of theAttorney Docket No.: GH0256WO bottom strand of the forward adapter and the 5-prime terminus of the bottom strand of the reverse adapter. In this way, the forward adapter (e.g., (P5) Illumina adapter) can be delivered to the 5- prime end of template molecules and the reverse adapter (e.g., (P7) Illumina adapter) is delivered to the 3-prime end of template molecules. Thus, the native polarity of input DNA molecules can be retained.

[0192] During the dual phosphorylation / ligation reaction, T4 Polynucleotide Kinase (PNK) can be used to prepare template DNA termini for ligation by phosphorylating 5-prime termini and dephosphorylating 3-prime termini. T4 PNK works on both ssDNA and dsDNA molecules and has no activity on the phosphorylation state of proteins. Simultaneously, the random nucleotides of the splint adapter can be annealed to the single-stranded template molecule. This creates a short, localized dsDNA molecule, enabling ligation of template to adapter with a ligase such as T4 DNA ligase, which has high ligation efficiency on dsDNA templates but low efficiency on ssDNA. After the single phosphorylation / ligation reaction is complete, the library DNA can be, e.g., purified and placed directly into standard NGS indexing PCR, compatible with both traditional single or dual index primers.

[0193] In some embodiments, following attachment of adapters, the nucleic acids are subject to amplification. The amplification can use, e.g., universal primers that recognize primer binding sites in the adapters.

[0194] In some embodiments, following attachment of adapters, the DNA is partitioned, comprising contacting the DNA with an agent that preferentially binds to nucleic acids bearing an epigenetic modification. The nucleic acids are partitioned into at least two subsamples differing in the extent to which the nucleic acids bear the modification from binding to the agents. For example, if the agent has affinity for nucleic acids bearing the modification, nucleic acids overrepresented in the modification (compared with median representation in the population) preferentially bind to the agent, whereas nucleic acids underrepresented for the modification do not bind or are more easily eluted from the agent. The nucleic acids can then be amplified from primers binding to the primer binding sites within the adapters. Partitioning may be performed instead before adapter attachment, in which case the adapters may comprise differential tags that include a component that identifies which partition a molecule occurred in.

[0195] In some embodiments, the nucleic acids are linked at both ends to Y-shaped adapters including primer binding sites and tags. The molecules are amplified.Attorney Docket No.: GH0256WO

[0196] Tagging DNA molecules is a procedure in which a tag is attached to or associated with the DNA molecules. Such tags can be molecules, such as nucleic acids, containing information that indicates a feature of the molecule with which the tag is associated. For example, molecules can bear a sample tag (which distinguishes molecules in one sample from those in a different sample) or a molecular tag / molecular barcode / barcode (which distinguishes different molecules from one another (in both unique and non-unique tagging scenarios). For methods that involve a partitioning step, a partition tag (which distinguishes molecules in one partition from those in a different partition) may be included. In some embodiments, adapters added to DNA molecules comprise tags. In certain embodiments, a tag can comprise one or a combination of barcodes. As used herein, the term “barcode” refers to a nucleic acid molecule having a particular nucleotide sequence, or to the nucleotide sequence, itself, depending on context. A barcode can have, for example, between 10 and 100 nucleotides. A collection of barcodes can have degenerate sequences or can have sequences having a certain hamming distance, as desired for the specific purpose. So, for example, a molecular barcode can be comprised of one barcode or a combination of two barcodes, each attached to different ends of a molecule. Additionally or alternatively, for different partitions and / or samples, different sets of molecular barcodes, or molecular tags can be used such that the barcodes serve as a molecular tag through their individual sequences and also serve to identify the partition and / or sample to which they correspond based the set of which they are a member.

[0197] In some embodiments, the EV marker binding molecule-oligonucleotide conjugates disclosed herein comprise a first portion comprising a universal sequence region, a second portion comprising a unique sequence region that identifies the second EV marker, and a third portion comprising a second universal sequence region, wherein the second universal sequence region comprises a double stranded portion and a single stranded portion. In some embodiments, the unique sequence region comprises a molecular tag. In some embodiments, the molecular tag is a molecular barcode. In some embodiments, the molecular barcode is used to determine the identity of the EV marker bound to the EV marker binding molecule-oligonucleotide conjugate.

[0198] In some embodiments, two or more partitions, e.g., each partition, is / are differentially tagged. Tags can be used to label the individual polynucleotide population partitions so as to correlate the tag (or tags) with a specific partition. Alternatively, tags can be used in embodiments that do not employ a partitioning step. In some embodiments, a single tag can be used to label a specific partition. In some embodiments, multiple different tags can be used to label a specificAttorney Docket No.: GH0256WO partition. In embodiments employing multiple different tags to label a specific partition, the set of tags used to label one partition can be readily differentiated for the set of tags used to label other partitions. In some embodiments, the tags may have additional functions, for example the tags can be used to index sample sources or used as unique molecular identifiers (which can be used to improve the quality of sequencing data by differentiating sequencing errors from mutations, for example as in Kinde et al., Proc Nat’l Acad Sci USA 108: 9530-9535 (2011), Kou et al., PLoS ONE,11 : eO 146638 (2016)) or used as non-unique molecule identifiers, for example as described in US Pat. No. 9,598,731. Similarly, in some embodiments, the tags may have additional functions, for example the tags can be used to index sample sources or used as non-unique molecular identifiers (which can be used to improve the quality of sequencing data by differentiating sequencing errors from mutations).

[0199] In some embodiments, partition tagging comprises tagging molecules in each partition with a partition tag. After re-combining partitions (e.g., to reduce the number of sequencing runs needed and avoid unnecessary cost) and sequencing molecules, the partition tags identify the source partition. In another embodiment, different partitions are tagged with different sets of molecular tags, e.g., comprised of a pair of barcodes. In this way, each molecular barcode indicates the source partition as well as being useful to distinguish molecules within a partition. For example, a first set of 35 barcodes can be used to tag molecules in a first partition, while a second set of 35 barcodes can be used tag molecules in a second partition.

[0200] In some embodiments, after partitioning and tagging with partition tags, the molecules may be pooled for sequencing in a single run. In some embodiments, a sample tag is added to the molecules, e.g., in a step subsequent to addition of partition tags and pooling. Sample tags can facilitate pooling material generated from multiple samples for sequencing in a single sequencing run.

[0201] Alternatively, in some embodiments, partition tags may be correlated to the sample as well as the partition. As a simple example, a first tag can indicate a first partition of a first sample; a second tag can indicate a second partition of the first sample; a third tag can indicate a first partition of a second sample; and a fourth tag can indicate a second partition of the second sample.

[0202] While tags may be attached to molecules already partitioned based on one or more characteristics, the final tagged molecules in the library may no longer possess that characteristic. For example, while single stranded DNA molecules may be partitioned and tagged, the final taggedAttorney Docket No.: GH0256WO molecules in the library are likely to be double stranded. Similarly, while DNA may be subject to partition based on different levels of methylation, in the final library, tagged molecules derived from these molecules are likely to be unmethylated. Accordingly, the tag attached to molecule in the library typically indicates the characteristic of the “parent molecule” from which the ultimate tagged molecule is derived, not necessarily to characteristic of the tagged molecule, itself.

[0203] As an example, barcodes 1, 2, 3, 4, etc. are used to tag and label molecules in the first partition; barcodes A, B, C, D, etc. are used to tag and label molecules in the second partition; and barcodes a, b, c, d, etc. are used to tag and label molecules in the third partition. Differentially tagged partitions can be pooled prior to sequencing. Differentially tagged partitions can be separately sequenced or sequenced together concurrently, e.g., in the same flow cell of an Illumina sequencer.

[0204] After sequencing, analysis of reads can be performed on a partition-by-partition level, as well as a whole DNA population level. Tags are used to sort reads from different partitions. Analysis can include in silico analysis to determine genetic and epigenetic variation (one or more of methylation, chromatin structure, etc.) using sequence information, genomic coordinates length, coverage, and / or copy number. In some embodiments, higher coverage can correlate with higher nucleosome occupancy in genomic region while lower coverage can correlate with lower nucleosome occupancy or a nucleosome depleted region (NDR).

[0205] Molecular tagging refers to a tagging practice that allows one to differentiate among DNA molecules from which sequence reads originated. Tagging strategies can be divided into unique tagging and non-unique tagging strategies. In unique tagging, all or substantially all of the molecules in a sample bear a different tag, so that reads can be assigned to original molecules based on tag information alone. Tags used in such methods are sometimes referred to as “unique tags”. In non-unique tagging, different molecules in the same sample can bear the same tag, so that other information in addition to tag information is used to assign a sequence read to an original molecule. Such information may include start and stop coordinate, coordinate to which the molecule maps, start or stop coordinate alone, etc. Tags used in such methods are sometimes referred to as “non-unique tags”. Accordingly, it is not necessary to uniquely tag every molecule in a sample. It suffices to uniquely tag molecules falling within an identifiable class within a sample. Thus, molecules in different identifiable families can bear the same tag without loss of information about the identity of the tagged molecule. «Attorney Docket No.: GH0256WO

[0206] In certain embodiments of non-unique tagging, the number of different tags used can be sufficient that there is a very high likelihood (e.g., at least 99%, at least 99.9%, at least 99.99% or at least 99.999% that all DNA molecules of a particular group bear a different tag. It is to be noted that when barcodes are used as tags, and when barcodes are attached, e.g., randomly, to both ends of a molecule, the combination of barcodes, together, can constitute a tag. This number, in term, is a function of the number of molecules falling into the calls. For example, the class may be all molecules mapping to the same start-stop position on a reference genome. The class may be all molecules mapping across a particular genetic locus, e.g., a particular base or a particular region (e.g., up to 100 bases or a gene or an exon of a gene). In certain embodiments, the number of different tags used to uniquely identify a number of molecules, z, in a class can be between any of 2*z, 3*z, 4*z, 5*z, 6*z, 7*z, 8*z, 9*z, 10*z, 11 *z, 12*z, 13*z, 14*z, 15*z, 16*z, 17*z, 18*z, 19*z, 20*z or 100*z (e.g., lower limit) and any of 100,000*z, 10,000*z, 1000*z or 100*z (e.g., upper limit).

[0207] For example, in a sample of about 5 ng to 30 ng of cell free DNA, one expects around 3000 molecules to map to a particular nucleotide coordinate, and between about 3 and 10 molecules having any start coordinate to share the same stop coordinate. Accordingly, about 50 to about 50,000 different tags (e.g., between about 6 and 220 barcode combinations) can suffice to uniquely tag all such molecules. To uniquely tag all 3000 molecules mapping across a nucleotide coordinate, about 1 million to about 20 million different tags would be required.

[0208] Generally, assignment of unique or non-unique tags barcodes in reactions follows methods and systems described by US patent applications 20010053519, 20030152490, 20110160078, and U.S. Pat. No. 6,582,908 and U.S. Pat. No. 7,537,898 and US Pat. No. 9,598,731.

[0209] Alternatively, in some embodiments, different nucleic acid molecules of a sample may be identified using only endogenous sequence information (e g., start and / or stop positions, subsequences of one or both ends of a sequence, and / or lengths). Tags can be linked to sample nucleic acids randomly or non-randomly.

[0210] In some embodiments, the assignment of unique molecular barcodes in reactions is performed using methods and systems described in Lim et al., Communications Biology. (2025)8: 1098, e.g., SPIDER-seq. In some such embodiments, amplicons are tagged with a pair of two unique molecular barcodes using primers that contain a barcode. Successive daughter strands synthesized through each round of PCR amplification are grouped into clusters (e.g., peer-to peerAttorney Docket No.: GH0256WO networks, as illustrated in Fig. 1c of Lim et al.) based on a chain of common unique barcodes between immediate parent and daughter strands. That is, strand synthesis events (with a synthesized strand as a template) involve copying one barcode from the template and include one new barcode from the primer, so each daughter strand shares a unique barcode with its parent. By clustering strands in this way, a consensus can be generated that reduces errors.

[0211] In some embodiments, the tagged nucleic acids are sequenced after loading into a microwell plate. The microwell plate can have 96, 384, or 1536 microwells. In some cases, they are introduced at an expected ratio of unique tags to microwells. For example, the unique tags may be loaded so that more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 50,000,000 or 1,000,000,000 unique tags are loaded per genome sample. In some cases, the unique tags may be loaded so that less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 50,000,000 or 1,000,000,000 unique tags are loaded per genome sample. In some cases, the average number of unique tags loaded per sample genome is less than, or greater than, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 50,000,000 or 1,000,000,000 unique tags per genome sample.

[0212] A preferred format uses 20-50 different tags (e.g., barcodes) ligated to both ends of target nucleic acids. For example, 35 different tags (e.g., barcodes) ligated to both ends of target molecules creating 35 x 35 permutations, which equals 1225 for 35 tags. Such numbers of tags are sufficient so that different molecules having the same start and stop points have a high probability (e.g., at least 94%, 99.5%, 99.99%, 99.999%) of receiving different combinations of tags. Other barcode combinations include any number between 10 and 500, e.g., about 15x15, about 35x35, about 75x75, about 100x100, about 250x250, about 500x500.

[0213] In some cases, unique tags may be predetermined or random or semi-random sequence oligonucleotides. In other cases, a plurality of barcodes may be used such that barcodes are not necessarily unique to one another in the plurality. In this example, barcodes may be ligated to individual molecules such that the combination of the barcode and the sequence it may be ligated to creates a unique sequence that may be individually tracked. As described herein, detection of non-unique barcodes in combination with sequence data of beginning (start) and end (stop) portions of sequence reads may allow assignment of a unique identity to a particular molecule. The length or number of base pairs, of an individual sequence read may also be used to assign aAttorney Docket No.: GH0256WO unique identity to such a molecule. As described herein, fragments from a single strand of nucleic acid having been assigned a unique identity, may thereby permit subsequent identification of fragments from the parent strand.F. Enriching / Capturing step

[0214] Some embodiments of the disclosed methods comprise enriching a sample for EVs comprising a first EV marker, which can involve isolating at least a portion of the extracellular vesicles in a sample from other components of the sample, thereby providing a sample enriched for extracellular vesicles.

[0215] In some embodiments, the enriching step comprises affinity purification, immunoprecipitation, size-exclusion chromatography, or centrifugation. In particular embodiments, the enriching comprises capturing extracellular vesicles in the sample using one or more extracellular vesicle-specific markers.

[0216] In some embodiments, the isolating comprises capturing extracellular vesicles in the sample using one or more binding molecules specific for one or more extracellular vesicle-specific markers. In some embodiments, the one or more binding molecules is bound to a solid support. In some such embodiments, the solid support comprises a bead or a substrate. Such methods allow for immobilization of the EV marker binding molecules.

[0217] In some embodiments, the methods provided herein comprise enriching for EVs comprising a first EV marker using an immobilized first EV marker binding molecule that binds to the first EV marker, thereby obtaining immobilized EVs.

[0218] In some embodiments, the immobilized first EV marker binding molecule comprises an antibody, a knottin, an aptamer, an affimer, an avimer, a nanobody, a DARPin, a monobody, or an affinity clamp (such as an antibody, a knottin, an aptamer, an affimer, an avimer, a nanobody, a DARPin, a monobody, or an affinity clamp specific for an EV-associated target molecule). In particular embodiments, the EV marker binding molecule comprises an antibody (such as an antibody specific for an EV-associated target molecule). In some embodiments, the immobilized first EV marker binding molecule is conjugated to a solid support (e.g., a bead or substrate).

[0219] In some embodiments, the first EV marker is selected from CEA, Mucl, PD-L1, CD9, CD3, CD151, CD81, CD63, ALIX, CD40, Hsp60, TSG1, and phosphatidylserine.

[0220] In some embodiments, the step of enriching occurs prior to a step of contacting the sample with a set of second EV marker binding molecule-oligonucleotide conjugates to generateAttorney Docket No.: GH0256WO complexes of second EV marker binding molecules bound to the immobilized EVs. In some embodiments, the step of enriching occurs prior to a step of partitioning extracellular vesicles in the sample into a plurality of compartments. In some embodiments, the step of enriching occurs after a step of partitioning the extracellular vesicles in the sample into a plurality of compartments.

[0221] In some embodiments, EVs in the sample that are bound to the one or more binding molecules (such as one or more immobilized EV marker binding molecules, such as one or more immobilized antibodies) are separated from other components of the sample, thereby providing separated bound extracellular vesicles. In some embodiments, the separating comprises affinity purification, immunoprecipitation, or a pull down assay. In some embodiments, the separating occurs prior to a step of partitioning, and at least a portion of the separated bound extracellular vesicles is subsequently partitioned. In some embodiments, the separating occurs after a step of partitioning.

[0222] The agents used to isolate EVs can be affinity agents, such as binding molecules (such as antibodies) with the desired specificity, natural binding partners or variants thereof (Bock et al., Nat Biotech 28: 1106-1114 (2010); Song et al., Nat Biotech 29: 68-72 (2011)). In some embodiments, EV-specific markers bound to an molecule (e.g., an immobilized EV marker binding molecule) used for affinity separation are subjected to a wash step. The wash step washes off EVs comprising EV-specific markers weakly bound to the affinity agent.

[0223] Methods disclosed herein can comprise isolating at least a portion of the extracellular vesicles in the sample from other components of the sample, such as from a first subsample. In some embodiments, the capturing comprises contacting the DNA with probes specific for the target regions. In some embodiments, the isolating comprises capturing extracellular vesicles in the sample using one or more extracellular vesicle-specific markers. Isolating, capturing, and separating EVs may be performed on any sample or subsample described herein using any suitable approach known in the art. In some embodiments, the binding molecules (such as antibodies) specific for one or more extracellular vesicle-specific markers comprise a capture moiety that facilitates the isolation, capture, and / or separation of the EVs.

[0224] As discussed above, EVs in a sample can be subject to a capture step, in which molecules having certain characteristics (such as one or more EV-specific markers) are captured and optionally used to separate EVs from other components of the sample, and / or are analyzed. EV capture can involve use of, e.g., an immobilized binding molecule (such as an antibody) labeledAttorney Docket No.: GH0256WO with a capture moiety, such as biotin or the other examples noted below. Binding molecules (such as binding molecules (such as antibodies) specific for one or more EV-specific markers) are combined with a sample under conditions that allow binding of the EV-specific markers with the binding agents. Then, captured EVs are isolated using the capture moiety. In some embodiments, binding molecules (such as antibodies) specific for one or more EV-specific markers can have higher and lower capture yields for different EV-specific markers.

[0225] Capture may be performed using any suitable approach known in the art. EV capture can involve use of an immobilized binding molecule (such as an antibody) labeled with a capture moiety, such as biotin or the other examples noted below. For example, a biotin capture moiety by bead-based streptavidin.

[0226] Capture moieties useful with the disclosed embodiments include, without limitation, biotin, avidin, streptavidin, a nucleic acid comprising a particular nucleotide sequence, digoxygenin, a histidine tag, an affinity tag, an immunoglobulin constant domain, a hapten recognized by a binding molecule (such as an antibody), and magnetically attractable particles. In some embodiments, an immunoglobulin constant domain may be bound using protein A, protein G, or a secondary antibody. In some embodiments, the secondary antibody comprises an anti-mouse secondary antibody. In some embodiments, the anti-mouse secondary antibody is a goat antimouse secondary antibody, rabbit anti-mouse secondary antibody, or a donkey anti-mouse secondary antibody. A capture moiety can be a member of a binding pair, such as biotin / streptavidin or hapten / antibody. In some embodiments, a capture moiety that is attached to a binding molecule (such as an antibody) specific for an EV-specific marker is captured by its binding pair which is attached to an isolatable moiety, such as a magnetically attractable particle or a large particle that can be sedimented through centrifugation. The capture moiety can be any type of molecule that allows affinity separation of EVs bearing the capture moiety from EVs lacking the capture moiety. Exemplary capture moieties are biotin that allows affinity separation by binding to streptavidin linked or linkable to a solid phase or an oligonucleotide, which allows affinity separation through binding to a complementary oligonucleotide linked or linkable to a solid phase.

[0227] In some embodiments, the binding molecules (such as antibodies) specific for one or more EV-specific markers comprise a capture moiety that facilitates the enrichment or capture of the EVs bound to the binding agents (such as antibodies). In some embodiments, the capture moietyAttorney Docket No.: GH0256WO is biotin. In some such embodiments, streptavidin attached to a solid support, such as magnetic beads, is used to bind to the biotin. Nonspecifically bound molecules or cell components that do not comprise a target EV-specific marker are washed away from the captured EVs. In some embodiments, EVs are then dissociated from the binding agents (such as antibodies) and eluted from the solid support using, e.g., washes or buffers. In some embodiments, the binding molecules (such as antibodies) are also eluted from the solid support by, e.g., disrupting the biotinstreptavidin interaction.

[0228] In some embodiments, at least a portion of the extracellular vesicles in a sample are isolated from other components of the sample using size-exclusion chromatography. Size exclusion chromatography (SEC) separates molecules based on their size, such as by fdtration through a gel matrix (such as in a column). A gel matrix for SEC can comprise beads (e.g., spherical beads) having pores of a specific size distribution appropriate for isolation of an analyte of interest (such as the EVs). Separation occurs when molecules or cell components (such as including EVs) of different sizes are included or excluded from the pores within the matrix. SEC can be used to efficiently separate EVs from circulating proteins with minimal vesicle alteration, including functionality or size, as compared to some other EV isolation methods.

[0229] In some embodiments, the immobilized EVs are released (e.g., released from the solid support) prior to a chromatography step. Releasing the immobilized EVs can mean removing the bound EVs from the surface on which they are immobilized (e.g., by cleaving a linker that connects the EV marker binding molecule to the solid support (e.g., the bead or substrate)).

[0230] In some embodiments, at least a portion of the extracellular vesicles in a sample are isolated from other components of the sample using centrifugation (i.e., extreme gravitational force). For example, EVs may be isolated using ultracentrifugation. Differential ultracentrifugation uses initial less extreme centrifugation steps to pellet cells and larger debris, before a final high gravitational force step (or steps) is used to pellet EVs from the remaining supernatant. Further refinement using density gradients may be included in ultracentrifugation workflows to improve the resolution of separation. SEC and centrifugation methods for isolating EVs from other components of a sample are known in the art (for further discussion, see Wang et al., J Transl Med, 2021, 19: 104, doi: 10.1186 / sl2967-021-02775-9; Gamez- Valero et al., Sci Rep, 2016, 6:33641, doi: 10.1038 / srep33641).5GAttorney Docket No.: GH0256WO

[0231] In some embodiments, the sample is partitioned into a plurality of subsamples. Tn some embodiments, the sample can be portioned into subsamples by repeating the step of contacting the sample with an immobilized first EV marker binding molecule (e.g., an immobilized EV marker binding antibody) one or more times.

[0232] In some such embodiments, the first EV marker binding molecule is immobilized on a solid support, such as a bead or substrate, to allow for separation of the bound EVs from nonbound EVs in the sample, thereby providing a sample comprising immobilized EVs. The sample comprising immobilized EVs is thereby enriched for EVs comprising the first EV marker. In some embodiments, each first EV marker is selected from CEA, Mucl, PD-L1, CD9, CD3, CD151, CD81, CD63, ALIX, CD40, Hsp60, TSG1, and phosphatidylserine.

[0233] In some such embodiments, the enrichment process is repeated one or more times with the remaining sample that was not previously enriched, thereby yielding a plurality of subsamples that are each enriched for a separate first EV marker. In some embodiments, each immobilized first EV marker binding molecule comprises an oligonucleotide, e.g., each immobilized first EV binding marker is an immobilized first EV binding maker-oligonucleotide conjugate, which can be amplified to add a molecular tag (e.g., a barcode) to identify the first EV marker. In some such embodiments, the molecular tags are used to identify each subsample of the plurality of subsamples to distinguish bound second EV marker binding molecule- oligonucleotide conjugates from each subsample.

[0234] In some embodiments, one or more samples comprising EVs are obtained from a subject. Each of these one or more samples can be optionally portioned into subsamples as described above. The one or more samples can be obtained from different tissues from the same subject. In some embodiments, each sample is contacted with an immobilized first EV marker binding molecule (e.g., an immobilized EV marker binding antibody). The first EV marker binding molecule is immobilized on a solid support, such as a bead or substrate, to allow for separation of the bound EVs from non-bound EVs in the sample, thereby providing a sample comprising immobilized EVs. Each sample comprising immobilized EVs is thereby enriched for EVs comprising the first EV marker. In some embodiments, each first EV marker is selected from CEA, Mucl, PD-L1, CD9, CD3, CD151, CD81, CD63, ALIX, CD40, Hsp60, TSG1, and phosphatidylserine.

[0235] In some such embodiments, the enrichment process is repeated for each sample. In some embodiments, each sample is enriched with a different immobilized first EV marker bindingAttorney Docket No.: GH0256WO molecule. In some such embodiments, the first EV marker is a tissue-specific and / or cell type specific marker. In some embodiments, each sample is enriched for the same first EV marker.

[0236] In some embodiments, each immobilized first EV marker binding molecule comprises an oligonucleotide, e.g., each immobilized first EV binding marker is an immobilized first EV binding maker-oligonucleotide conjugate, which can be amplified to add a molecular tag (e.g., a barcode) to identify the first EV marker and / or the tissue of origin of the sample. In some such embodiments, the molecular tags are used to identify each sample of the plurality of samples to distinguish bound second EV marker binding molecule-oligonucleotide conjugates from each sample.

[0237] Methods disclosed herein can comprise enriching, capturing, or isolating post- translationally modified proteins and / or target proteins, and / or enriching, capturing, or isolating DNA, such as cfDNA target regions. In some embodiments, the capturing comprises contacting the post-translationally modified proteins and / or target proteins with binding molecules specific for a PTM and / or the target protein, and / or contacting the DNA with probes specific for target regions. Enrichment or capture may be performed on any sample or subsample described herein using any suitable approach known in the art.

[0238] In some embodiments, the binding molecules specific for a PTM or target protein or the probes specific for DNA target regions comprise a capture moiety that facilitates the enrichment or capture of target proteins or the DNA hybridized to the probes, respectively. In some embodiments, the capture moiety is biotin. In some such embodiments, streptavidin attached to a solid support, such as magnetic beads, is used to bind to the biotin. In some embodiments, nonspecifically bound material (e.g., DNA that does not comprise a target region) is washed away from the captured material. In some embodiments, captured material is then dissociated from the probes and eluted from the solid support using salt washes or buffers comprising another DNA denaturing agent. In some embodiments, the binding molecules and / or probes are also eluted from the solid support by, e.g., disrupting the biotin-streptavidin interaction. In some embodiments, captured DNA and / or oligonucleotide labels is / are amplified following elution from the solid support. In some such embodiments, DNA comprising adapters is amplified using PCR primers that anneal to the adapters. In some embodiments, captured DNA is amplified while attached to the solid support. In some such embodiments, the amplification comprises use of a PCR primer that anneals to a sequence within an adapter and a PCR primer that anneals to a sequence within a probe annealed to the target region of the DNA.Attorney Docket No.: GH0256WO

[0239] In some embodiments, the methods herein comprise enriching for or capturing DNA comprising epigenetic and / or sequence-variable target regions. Such regions may be captured from an aliquot of a sample (e.g., a sample that has undergone attachment of adapters and amplification), while the step of partitioning the DNA with an agent that recognizes methyl cytosine is performed on a separate aliquot of the sample. Enriching for or capturing DNA comprising epigenetic and / or sequence-variable target regions may comprise contacting the DNA with a first or second set of target-specific probes. Such target-specific probes may have any of the features described herein for sets of target-specific probes, including but not limited to in the embodiments set forth above and the sections relating to probes below. Capturing may be performed on one or more subsamples prepared during methods disclosed herein. In some embodiments, DNA is captured from the first subsample or the second subsample, e.g., the first subsample and the second subsample. In some embodiments, the subsamples are differentially tagged (e.g., as described herein) and then pooled before undergoing capture. Exemplary methods for capturing DNA comprising epigenetic and / or sequence-variable target regions can be found in, e.g., WO 2020 / 160414, which is hereby incorporated by reference.

[0240] The capturing step may be performed using conditions suitable for specific nucleic acid hybridization, which generally depend to some extent on features of the probes such as length, base composition, etc. Those skilled in the art will be familiar with appropriate conditions given general knowledge in the art regarding nucleic acid hybridization. In some embodiments, complexes of target-specific probes and DNA are formed.

[0241] In some embodiments, methods described herein comprise capturing a plurality of sets of target regions of cfDNA obtained from a subject. The target regions may comprise differences depending on whether they originated from a tumor or from healthy cells or from a certain cell type. The capturing step produces a captured set of cfDNA molecules. In some embodiments, cfDNA molecules corresponding to a sequence-variable target region set are captured at a greater capture yield in the captured set of cfDNA molecules than cfDNA molecules corresponding to an epigenetic target region set. In some embodiments, a method described herein comprises contacting cfDNA obtained from a subject with a set of target-specific probes, wherein the set of target-specific probes is configured to capture cfDNA corresponding to the sequence-variable target region set at a greater capture yield than cfDNA corresponding to the epigenetic target regionAttorney Docket No.: GH0256WO set. For additional discussion of capturing steps, capture yields, and related aspects, see W02020 / 160414, which is incorporated herein by reference for all purposes.

[0242] It can be beneficial to capture cfDNA corresponding to the sequence-variable target region set at a greater capture yield than cfDNA corresponding to the epigenetic target region set because a greater depth of sequencing may be necessary to analyze the sequence-variable target regions with sufficient confidence or accuracy than may be necessary to analyze the epigenetic target regions. The volume of data needed to determine fragmentation patterns (e.g., to test for perturbation of transcription start sites or CTCF binding sites) or fragment abundance (e.g., in hypermethylated and hypomethylated partitions) is generally less than the volume of data needed to determine the presence or absence of cancer-related sequence mutations. Capturing the target region sets at different yields can facilitate sequencing the target regions to different depths of sequencing in the same sequencing run (e.g., using a pooled mixture and / or in the same sequencing cell).

[0243] In some embodiments, the DNA is amplified. In some embodiments, amplification is performed before the capturing step. In some embodiments, amplification is performed after the capturing step. In some embodiments, amplification is performed before and after the capturing step. In various embodiments, the methods further comprise sequencing the captured DNA, e.g., to different degrees of sequencing depth for the epigenetic and sequence-variable target region sets, consistent with the discussion herein.

[0244] In some embodiments, a capturing step is performed with probes for a sequence-variable target region set and probes for an epigenetic target region set in the same vessel at the same time, e.g., the probes for the sequence-variable and epigenetic target region sets are in the same composition. This approach provides a relatively streamlined workflow.

[0245] In some embodiments, adapters are included in the DNA as described herein. In some embodiments, tags, which may be or include barcodes, are included in the DNA. In some embodiments, such tags are included in adapters. Tags can facilitate identification of the origin of a nucleic acid. For example, barcodes can be used to allow the origin (e.g., subject) whence the DNA came to be identified following pooling of a plurality of samples for parallel sequencing. This may be done concurrently with an amplification procedure, e.g., by providing the barcodes in a 5’ portion of a primer, e.g., as described herein. In some embodiments, adapters and tags / barcodes are provided by the same primer or primer set. For example, the barcode may beAttorney Docket No.: GH0256WO located 3’ of the adapter and 5’ of the target-hybridizing portion of the primer. Alternatively, barcodes can be added by other approaches, such as ligation, optionally together with adapters in the same ligation substrate.

[0246] Additional details regarding amplification, tags, and barcodes are discussed herein, which can be combined to the extent practicable with any of these embodiments.G. Detecting

[0247] Embodiments of the disclosed methods comprise enriching for EVs from a sample obtained from a subject using a first immobilized EV marker binding molecule and then detecting a plurality of EV-associated target molecules in the sample using a set of second EV marker binding molecule-oligonucleotide conjugates. In some embodiments, the methods further comprise detecting a level of each of a plurality of second EV-associated target molecules. In some such embodiments, the method comprises quantifying each of the plurality of EV-associated target molecules, such within and / or on an EV. In some embodiments, the EV is in a partitioned compartment, such as a droplet. For example, use of the disclosed methods in particular examples permits detection of oligonucleotides of a plurality of EV marker binding agent-oligonucleotide conjugates.

[0248] In some embodiments, the methods provided herein comprise (a) obtaining an EV- containing sample; (b) enriching for EVs comprising a first EV marker using an immobilized first EV marker binding molecule that binds to the first EV marker, thereby obtaining immobilized EVs; (c) contacting the immobilized EVs with a set of second EV marker binding molecule- oligonucleotide conjugates to generate complexes of second EV marker binding molecules bound to the immobilized EVs; (d) removing unbound second EV marker binding molecule- oligonucleotide conjugates from the complexes of second EV marker binding molecules bound to the immobilized EVs; and (e) detecting the second EV marker binding molecules bound to the immobilized EVs.

[0249] In some embodiments, step (a) is repeated one or more times on the sample to provide a plurality of subsamples. In some embodiments, step (a) is performed for each of plurality of samples obtained from the subject (e.g., from different tissues from the same subject). In some embodiments, steps (b)-(d) are repeated for each of the plurality of samples or subsamples.

[0250] In some embodiments, the methods provided herein comprise (a) obtaining an EV- containing sample; (b) enriching for EVs comprising a first EV marker using an immobilized firstAttorney Docket No.: GH0256WOEV marker binding molecule that binds to the first EV marker, thereby obtaining immobilized EVs; (c) contacting the immobilized EVs with a set of second EV marker binding molecule- oligonucleotide conjugates to generate complexes of second EV marker binding molecules bound to the immobilized EVs; (d) releasing the immobilized EVs, thereby providing released EVs; (e) removing unbound second EV marker binding molecule-oligonucleotide conjugates from the complexes of second EV marker binding molecules bound to the released EVs using chromatographic purification; and (f) detecting the second EV marker binding molecules bound to the released EVs. In some such embodiments, the immobilized EVs are released (e.g., released from the solid support) prior to a chromatography step. Releasing the immobilized EVs can mean removing the bound EVs from the surface on which they are immobilized (e.g., by cleaving a linker that connects the EV marker binding molecule to the solid support (e.g., the bead or substrate)).

[0251] In some embodiments, step (a) is repeated one or more times on the sample to provide a plurality of subsamples. In some embodiments, step (a) is performed for each of plurality of samples obtained from the subject (e.g., from different tissues from the same subject). In some embodiments, steps (b)-(e) are repeated for each of the plurality of samples or subsamples.

[0252] In some embodiments, the set of second EV marker binding molecule-oligonucleotide conjugates comprises 2-100 EV marker binding molecule-oligonucleotide conjugates. In some embodiments, the set of second EV marker binding molecule-oligonucleotide conjugates comprises 2-100, 10-50, 20-40, 25-35, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more than 60 EV marker binding molecule-oligonucleotide conjugates.

[0253] In some embodiments, the set of second EV marker binding molecule-oligonucleotide conjugates does not comprise an EV marker binding molecule-oligonucleotide conjugate that binds the same EV marker as the first immobilized EV marker binding molecule. In some embodiments, the set of second EV marker binding molecule-oligonucleotide conjugates comprises an EV marker binding molecule-oligonucleotide conjugate that binds the same EV marker as the first immobilized EV marker binding molecule.

[0254] In some embodiments, one or more of the plurality, the majority of the plurality, or all of the plurality of second EV marker binding molecules comprise an antibody, a knottin, an aptamer, an affimer, an avimer, a nanobody, a DARPin, a monobody, or an affinity clamp (such as an antibody, a knottin, an aptamer, an affimer, an avimer, a nanobody, a DARPin, a monobody, or anAttorney Docket No.: GH0256WO affinity clamp specific for an EV-associated target molecule). Tn particular embodiments, the EV marker binding molecule comprises an antibody (such as an antibody specific for an EV-associated target molecule).

[0255] In some embodiments, each second EV marker binding molecule-oligonucleotide conjugate further comprises a first portion comprising a universal sequence region, a second portion comprising a unique sequence region that identifies an EV marker, and a third portion comprising a second universal sequence region. In some embodiments, the second universal sequence region comprises a double stranded portion and a single stranded portion.

[0256] In some embodiments, the second universal sequence region comprises the 3’ end of the first oligonucleotide. In some embodiments, the single stranded portion of the second universal sequence region is located 3’ of the double stranded portion of the second universal sequence region. In some embodiments, the unique sequence region is distal to the single stranded portion of the second universal sequence region relative to the double stranded portion of the second universal sequence region. In some embodiments, the unique sequence region comprises a molecular tag such as a molecular barcode.

[0257] In some embodiments, the sample obtained from the subject is partitioned into a plurality of subsamples. In some embodiments, the sample can be portioned into subsamples by repeating the step of contacting the sample with an immobilized first EV marker binding molecule (e.g., an immobilized EV marker binding antibody) one or more times.

[0258] In some such embodiments, the enrichment process is repeated one or more times with the remaining sample that was not previously enriched, thereby yielding a plurality of subsamples that are each enriched for a separate first EV marker.

[0259] In some embodiments, one or more samples comprising EVs are obtained from a subject. In some embodiments, the one or more samples are obtained from different tissues from the same subject. In some embodiments, each sample is contacted with an immobilized first EV marker binding molecule (e.g., an immobilized EV marker binding antibody).

[0260] In some embodiments, each immobilized first EV marker binding molecule comprises an oligonucleotide, e.g., each immobilized first EV binding marker is an immobilized first EV binding maker-oligonucleotide conjugate, which can be amplified to add a molecular tag (e.g., a barcode) to identify the first EV marker. In some such embodiments, the molecular tags are used to identifyAttomey Docket No.: GH0256WO each sample or subsample of the plurality of samples or subsamples to distinguish bound second EV marker binding molecule-oligonucleotide conjugates from each subsample.

[0261] In some embodiments, each sample or sub sample of the plurality of samples or sub samples is contacted with a set of second EV marker binding molecule-oligonucleotide conjugates (e.g., antibody-oligonucleotide conjugates) to generate complexes of second EV marker binding molecules bound to the immobilized EVs.

[0262] In some embodiments, second EV marker binding molecule-oligonucleotide conjugates are removed from each sample or subsample comprising immobilized EVs (e g., by a wash step) to yield a plurality of purified samples or subsamples comprising immobilized EVs bound to second EV marker binding-oligonucleotide conjugates.

[0263] In some embodiments, the bound second EV marker binding molecule-oligonucleotide conjugates from each sample or subsample are detected by methods known in the art for the detection of nucleic acids (e.g., by high multiplex PCR, digital PCR, quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), or sequencing). In some embodiments, the detection step comprises an amplification step (e.g., a PCR step) and a sequencing step (e.g., next generation sequencing (NGS)). In some embodiments, molecular tags such as barcodes, sample indices, and / or adapters are added during the amplification step to enable multiplexing of the sequencing step. In some such embodiments, the molecular tags are used to identify each sample or subsample of the plurality of samples or subsamples to distinguish bound second EV marker binding molecule-oligonucleotide conjugates from each subsample.

[0264] In certain embodiments wherein the plurality of EV-associated target nucleic acids comprise RNA, the detecting comprises RT-PCR, such as prior to digital PCR (such as droplet digital PCR) or sequencing (such as next generation sequencing). EV dissociation or lysis within each droplet, such as to release EV-associated nucleic acids from an internal portion of an EV (e.g., prior to an amplification or detection step), may be accomplished using methods known in the art, such as enzymatic dissociation or lysis.

[0265] In some embodiments, the immobilized first EV marker binding molecule is conjugated to an solid support. In some embodiments, the immobilized first EV marker binding molecule comprises an oligonucleotide comprising a capture nucleotide sequence. In some embodiments, the capture nucleotide sequence is linked to the solid support. In some embodiments, the capture nucleotide sequence can hybridize with a universal region of an EV marker binding molecule-Attorney Docket No.: GH0256WO oligonucleotide conjugate and thereby participate in proximity extension reactions. In some embodiments, the proximity extension reaction yields an extension product. In some embodiments, the extension product is amplified by PCR.

[0266] In some embodiments, the first EV marker is selected from CEA, Mucl, PD-L1, CD9, CD3, CD151, CD81, CD63, ALIX, CD40, Hsp60, TSG1, and phosphatidylserine. In some embodiments, the first EV marker is a tissue-specific marker and / or a cell type marker.

[0267] In some embodiments, the second EV marker binding molecules bind to one or more of more of ALIX, CD40, TGS1, phosphatidylserine, Mucl, CD3, CD147, CD151, carcinoembryonic antigen (CEA), CD66a, CD326 (also known as epithelial cell adhesion molecule, EpCAM), CD49b, heat shock protein 60 (Hsp60), copine-3 (CPNE3), folate receptor alpha (FRa), CD24, vascular endothelial growth factor (VEGF), prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), epidermal growth factor receptor (EGFR), Glypican-3 (GPC-3), Glypican-1 (GPC-1), CD63, CD9, programmed death ligand 1 (PD-L1), leucine-rich alpha-2- glycoprotein 1 (LRG1), Epstein-Barr virus latent membrane protein 1 (LMP1), Epstein-Barr virus BamHI- A rightward frame 1 (BARF1), matrix metalloproteinase 9 (MMP9), ceruloplasmin (CP), Dickkopf-related protein 4 (DKK4), carbonic anhydrase 9 (CAIX), tumor-associated calcium signal transducer 2 (TACSTD2), CD36, CD44, Claudin 4, LI cell adhesion molecule (LI CAM), CD24, A disintegrin and metalloproteinase domain-containing protein 10 (ADAMI 0), extracellular matrix metalloproteinase inducer (EMMPRIN), transforming growth factor P-1 (TGFP-1), melanoma antigen 3 / 6 (MAGE-3 / 6), CD37, CD53, CD81, CD47, CD26, CD196, CD274, CD133, and / or Ephrin A2. In some embodiments, the set of second EV marker binding molecule-oligonucleotide conjugates also comprises one or more control binding molecule- oligonucleotide conjugates (e.g., an isotype control antibody-oligonucleotide conjugate).

[0268] In some embodiments, the second EV marker binding molecules bind to 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, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 or more, 37 or more, 38 or more, 39 or more, 40 or more, 41 or more, 42 or more, 43 or more, 44 or more, 45 or more, 46 or more, 47 or more, 48 or more, 49 or more, 50 or more, or each of ALIX, CD40, TGS1, phosphatidyl serine, Mucl, CD3, CD147, CD151,Attorney Docket No.: GH0256WO carcinoembryonic antigen (CEA), CD66a, CD326 (also known as epithelial cell adhesion molecule, EpCAM), CD49b, heat shock protein 60 (Hsp60), copine-3 (CPNE3), folate receptor alpha (FRa), CD24, vascular endothelial growth factor (VEGF), prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), epidermal growth factor receptor (EGFR), Glypican- 3 (GPC-3), Glypican-1 (GPC-1), CD63, CD9, programmed death ligand 1 (PD-L1), leucine-rich alpha-2-glycoprotein 1 (LRG1), Epstein-Barr virus latent membrane protein 1 (LMP1), Epstein- Barr virus BamHI-A rightward frame 1 (BARF1), matrix metalloproteinase 9 (MMP9), ceruloplasmin (CP), Dickkopf-related protein 4 (DKK4), carbonic anhydrase 9 (CAIX), tumor- associated calcium signal transducer 2 (TACSTD2), CD36, CD44, Claudin 4, LI cell adhesion molecule (LI CAM), CD24, A disintegrin and metalloproteinase domain-containing protein 10 (ADAM 10), extracellular matrix metalloproteinase inducer (EMMPRIN), transforming growth factor p-1 (TGFP-1), melanoma antigen 3 / 6 (MAGE-3 / 6), CD37, CD53, CD81, CD47, CD26, CD196, CD274, CD133, and / or Ephrin A2. In some embodiments, the set of second EV marker binding molecule-oligonucleotide conjugates also comprises one or more control binding molecule-oligonucleotide conjugates (e.g., an isotype control antibody-oligonucleotide conjugate).1. Digital PCR

[0269] In some embodiments, EVs are distributed among a plurality of compartments such as a plurality of droplets, and a detection step comprises digital PCR, such as droplet digital PCR. A droplet digital PCR (ddPCR) system can use, e g., an immiscible fluid in oil to generate a plurality of droplets, such as submicroliter droplets. In some cases, a ddPCR system can generate thousands to millions of such droplets. In such embodiments, EVs from a sample are encapsulated randomly inside the droplets, which can serve as mini reaction chambers. In some embodiments, a plurality of droplets is generated such that only a single extracellular vesicle is present in at least a portion of the plurality of droplets.

[0270] The digital droplet PCR reaction in a ddPCR workflow may be prepared in a tube. A ddPCR mix can be added to a sample and then partitioned into individual droplets using a droplet generator. The emulsion is collected, e.g., in a vial, and PCR is performed. EV dissociation or lysis within each droplet, such as to release EV-associated nucleic acids from an internal portion of an EV, may be accomplished using methods known in the art, such as enzymatic dissociation or lysis. A digital PCR instrument can separately amplify nucleic acids present in the individual droplets,6GAttorney Docket No.: GH0256WO such as oligonucleotides of binding agent-oligonucleotide conjugates (such as antibody- oligonucleotide conjugates) bound to an EV, and / or endogenous EV-associated nucleic acids. The sample can be processed using a flow cytometer where droplets are fluorescently read one by one as they pass in front of a laser excitation source. The number of copies of DNA or cDNA template in the initial reaction can be determined, e.g., using Poisson statistics. Discussions of droplet digital PCR can be found in Lai et al., bioRxiv, 2023.10.09.561546, doi: 10.1101 / 2023.10.09.561546; and Yap et al., BioTechniques, 2020, 69(2), 99-107, doi: 10.2144 / btn-2020-0028.2. Sequencing

[0271] In some embodiments, the detection step comprises sequencing (such as using next generation sequencing) oligonucleotides of the plurality of EV binding agent-oligonucleotide conjugates (such as antibody-oligonucleotide conjugates) and / or EV-associated target molecules comprising DNA and / or RNA, such as described in detail elsewhere herein. In some embodiments, sequence reads are obtained and analyzed in silico. After sequencing, analysis of reads, such as to identify oligonucleotides and / or detect genetic variants in EV-associated nucleic acids, can be performed on a compartment-by-compartment (e.g., droplet-by-droplet) level, as well as a whole nucleic acid population level. In some embodiments, tags (such as comprising barcodes) can be used to sort reads from different compartments. For example, analysis can include in silico analysis to determine genetic variants, such as copy number variations (CNVs), single nucleotide variations (SNVs), insertions / deletions (indels), and / or fusions in a plurality of EV-associated target molecules comprising nucleic acids in each partition. Analyzing EV-associated DNA may comprise detecting or quantifying DNA of interest associated with an EV present within a compartment. Analyzing EV-associated DNA can comprise detecting genetic variants associated with an EV present within a compartment.

[0272] Exemplary sequencing techniques for use in the disclosed methods can include, for example, Sanger sequencing, high-throughput sequencing, pyrosequencing, sequencing-by- synthesis, long-read sequencing (also known as single-molecule sequencing or third generation sequencing), nanopore sequencing (a type of long-read sequencing), 5-letter sequencing or 6-letter sequencing, semiconductor sequencing, sequencing-by-ligation, sequencing-by-hybridization, Digital Gene Expression (Helicos), Next generation sequencing (NGS), Single Molecule Sequencing by Synthesis (SMSS) (Helicos), massively-parallel sequencing, Clonal Single Molecule Array (Solexa), shotgun sequencing, Ion Torrent, Oxford Nanopore, Roche Genia,Attorney Docket No.: GH0256WOMaxim-Gilbert sequencing, primer walking, and sequencing using PacBio, SOLiD, Ion Torrent, or Nanopore platforms. Sequencing reactions can be performed in a variety of sample processing units, which may include multiple lanes, multiple channels, multiple wells, or other means of processing multiple sample sets substantially simultaneously. Sample processing units can also include multiple sample chambers to enable processing of multiple runs simultaneously.

[0273] In some embodiments, sequencing comprises detecting and / or distinguishing unmodified and modified nucleobases. For example, long-read sequencing (also referred to herein as third generation sequencing) methods include those that can generate longer sequencing reads, such as reads in excess of 10 kilobases, as compared to short-read sequencing methods, which generally produce reads of up to about 600 bases in length. Compared to short reads, long reads can improve de novo assembly, transcript isoform identification, and detection and / or mapping of structural variants. Furthermore, long-read sequencing of native DNA or RNA molecules reduces amplification bias and preserves base modifications, such as methylation status. Long-read sequencing technologies useful herein can include any suitable long-read sequencing methods, including, but not limited to, Pacific Biosciences (PacBio) single-molecule real-time (SMRT) sequencing, Oxford Nanopore Technologies (ONT) nanopore sequencing, and synthetic long-read sequencing approaches, such as linked reads, proximity ligation strategies, and optical mapping. Synthetic long-read approaches comprise assembly of short reads from the same DNA molecule to generate synthetic long reads, and may be used in conjunction with “true” long-read sequencing technologies, such as SMRT and nanopore sequencing methods.

[0274] Single-molecule real-time (SMRT) sequencing can facilitate direct detection of, e.g., 5- methylcytosine and 5-hydroxymethylcytosine as well as unmodified cytosine. (Weirather JL, et al., “Comprehensive comparison of Pacific Biosciences and Oxford Nanopore Technologies and their applications to transcriptome analysis,” FlOOOResearch, 6: 100, 2017). Whereas nextgeneration sequencing methods detect augmented signals from a clonal population of amplified DNA fragments, SMRT sequencing captures a single DNA molecule, maintaining base modification during sequencing. The error rate of raw PacBio SMRT sequencing-generated data is about 13-15%, as the signal -to-noise ratio from single DNA molecules not high. To increase accuracy, this platform uses a circular DNA template by ligating hairpin adapters to both ends of target double-stranded DNA. As the polymerase repeatedly traverses and replicates the circular molecule, the DNA template is sequenced multiple times to generate a continuous long readAttorney Docket No.: GH0256WO(CLR). The CLR can be split into multiple reads (“subreads”) by removing adapter sequences, and multiple subreads generate circular consensus sequence (“CCS”) reads with higher accuracy. The average length of a CLR is >10 kb and up to 60 kb, with length depending on the polymerase lifetime. Thus, the length and accuracy of CCS reads depends on the fragment sizes. PacBio sequencing has been utilized for genome (e.g., de novo assembly, detection of structural variants and haplotyping) and transcriptome (e.g., gene isoform reconstruction and novel gene / isoform discovery) studies.

[0275] SMRT sequencing relies on sequencing-by-synthesis, where the sequence of a circular DNA template is determined from the succession of fluorescence pulses, each resulting from the addition of one labelled nucleotide by a polymerase fixed to the bottom of a well. Base modifications do not affect the base-called sequence, but they affect the kinetics of the polymerase. By considering the inter-pulse duration (IPD), base modifications can be inferred from the comparison of a modified template to an in silico model or an unmodified template. Such methods can therefore use the pulse width of a signal from sequencing bases, the interpulse duration (IPD) of bases, and the identity of the bases in order to detect a modification in a base or in a neighboring base. (See e.g., Weirather et al., FlOOOResearch, 6: 100, 2017.) SMRT sequencing can thus be used to detect base modifications such as 5-caC, 4mC, 5mC, 5hmC, 6mA, and 8oxoG (Gouil & Keniry Essays in Biochemistry (2019) 63 639-648). Accordingly, in some embodiments, the sequencing comprises SMRT sequencing.

[0276] Some sequencing reactions involve use of an enzyme to control passage of a nucleic acid through a nanopore, and in such cases reaction data can include both kinetics and other behavior of the enzyme and fluctuations in current through the nanopore. For example, ratchet proteins, helicases, or motor proteins can be used to push or pull a nucleic acid molecule through a hole in a biological or synthetic membrane. The kinetics of these proteins can vary depending on the sequence context of a nucleic acid on which they are acting. For example, they may slow down or pause at a modified base, and this behavior, captured as a part of the reaction data, is indicative of the presence of the modified base even where the modified base is not within the sensing portion of the nanopore.

[0277] One example of a nanopore-based single molecule sequencing system is that commercialized by Oxford Nanopore Technologies (ONT). (Weirather JL, et al., FlOOOResearch, 6: 100, 2017). ONT directly sequences a native single- stranded DNA (ssDNA) molecule byAttomey Docket No.: GH0256WO measuring characteristic current changes as the bases are threaded through the nanopore by a molecular motor protein. ONT uses a hairpin library structure similar to the PacBio circular DNA template: the DNA template and its complement are bound by a hairpin adapter. Therefore, the DNA template passes through the nanopore, followed by a hairpin and finally the complement. The raw read can be split into two “ID” reads (“template” and “complement”) by removing the adapter. The consensus sequence of two “ID” reads is a “2D” read with a higher accuracy.

[0278] Nanopore sequencing can be used to detect base modifications including 5-caC, 5mC, 5hmC, 6mA, BrdU, FldU, IdU, and EdU (see e.g., Gouil & Keniry Essays in Biochemistry (2019) 63 639-648; Kutyavin, Biochemistry (2008), 47, 51, 13666-1367; Muller et al., Nature Methods (2019), volume 16, pages 429-436; Hennion et al., Genome Biology (2020), volume 21, Article number: 125). Accordingly, in some embodiments, the sequencing comprises nanopore sequencing.

[0279] In some embodiments, the sequencing comprises single cell sequencing (See, Kashima et al., Exp Mol Med, 2020, 52: 1419-1427, doi: 10.1038 / sl2276-020-00499-2; Luo et al., Small Methods, 2022, 6(1 l):e2200881, doi: 10.1002 / smtd.202200881). Single-cell DNA and / or RNA sequencing can be used with the disclosed methods to sequence EV-associated target molecules comprising DNA and / or RNA by amplifying total nucleic acids associated with the EV of a partitioned compartment and / or oligonucleotides of binding agent-oligonucleotide conjugates (such as antibody-oligonucleotide conjugates) bound to the EV, and then applying next-generation DNA sequencing. Tags (such as tags comprising barcodes) may be added to EV-associated nucleic acid molecules before amplification, to mark a sequence read as coming from a specific starting EV. Amplified nucleic acids can also be labeled with barcodes (such as using singlecell combinatorial indexing RNA sequencing or split-pool ligation-based transcriptome sequencing). EV dissociation within each compartment (e.g., droplet) or lysis may be accomplished using methods known in the art, such as enzymatic dissociation or lysis.3. Proximity Extension Assay; Proximity Ligation Assay

[0280] In some embodiments, the immobilized first EV marker binding molecule is conjugated to an solid support. In some embodiments, the immobilized first EV marker binding molecule comprises an oligonucleotide comprising a capture nucleotide sequence. In some embodiments, the capture nucleotide sequence is linked to the solid support. In some embodiments, the capture nucleotide sequence can hybridize with a universal region of an EV marker binding molecule-Attorney Docket No.: GH0256WO oligonucleotide conjugate and thereby participate in proximity extension reactions. In some embodiments, the proximity extension reaction yields an extension product. In some embodiments, the extension product is amplified by PCR.

[0281] In some embodiments, the detecting comprises a proximity extension assay. In a proximity extension assay, one or more EV marker binding molecules (such as first and second EV marker binding molecules, such as antibodies) that target EV-associated target molecules in close proximity (such as an EV-associated protein, such as an EV-associated surface protein), or an EV- specific marker (such as an EV-specific target protein, such as one or more of ALIX, CD40, TGS1, phosphatidyl serine, Muci, CD3, CD147, CD151, carcinoembryonic antigen (CEA), CD66a, CD326 (also known as epithelial cell adhesion molecule, EpCAM), CD49b, heat shock protein 60 (Hsp60), copine-3 (CPNE3), folate receptor alpha (FRa), CD24, vascular endothelial growth factor (VEGF), prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), epidermal growth factor receptor (EGFR), Glypican-3 (GPC-3), Glypican-1 (GPC-1), CD63, CD9, programmed death ligand 1 (PD-L1), leucine-rich alpha-2-glycoprotein 1 (LRG1), Epstein-Barr virus latent membrane protein 1 (LMP1), Epstein-Barr virus BamHI-A rightward frame 1 (BARF1), matrix metalloproteinase 9 (MMP9), ceruloplasmin (CP), Dickkopf-related protein 4 (DKK4), carbonic anhydrase 9 (CAIX), tumor-associated calcium signal transducer 2 (TACSTD2), CD36, CD44, Claudin 4, LI cell adhesion molecule (L1CAM), CD24, A disintegrin and metalloproteinase domain-containing protein 10 (ADAM 10), extracellular matrix metalloproteinase inducer (EMMPRIN), transforming growth factor P-1 (TGFP-1), melanoma antigen 3 / 6 (MAGE-3 / 6), CD37, CD53, CD81, CD47, CD26, CD196, CD274, CD133, and / or Ephrin A2) and an EV-associated target molecule (such as an EV-associated protein, such as an EV-associated surface protein), are conjugated to oligonucleotides that comprise complementary hybridization sequences which are optionally 3’ of a tag (e.g., a molecular barcode, which identifies the type of binding molecule with which the oligonucleotide was associated (e.g., a molecular barcode comprising a sequence that is unique to the type of binding agent (such as the type of antibody), such as a sequence that is unique to a binding agent (such as an antibody) that is specific for a particular EV-associated target molecule or EV-specific marker) and may provide additional information, e.g., regarding the sample). The tags may have any of the features described elsewhere herein with respect to tags. When the oligonucleotides are in proximity (as occurs when the binding agents (such as the antibodies) are bound to EV-associated targetAttorney Docket No.: GH0256WO molecules in close proximity, or the EV-associated target molecule and an EV-specific marker), the hybridization sequences can hybridize to each other, forming a substrate for extension by a DNA polymerase. The extended product can then be detected (e.g., by sequencing or PCR, which may follow an amplification step), thus indicating co-localization of the two or more EV marker binding molecules. Where the presence, absence, or level of a plurality of target EV-associated target molecules is being detected, the assay may be multiplexed.

[0282] In some embodiments, the detecting comprises a proximity ligation assay. In a proximity ligation assay, one or more EV marker binding molecules (such as first and second EV marker binding molecules, such as antibodies) that target the same EV-associated target molecule (such as an EV-associated protein, such as an EV-associated surface protein), or an EV-specific marker (such as an EV-specific target protein) and an EV-associated target molecule (such as an EV- associated protein, such as an EV-associated surface protein, such as one or more of one or more of ALIX, CD40, TGS1, phosphatidylserine, Mucl, CD3, CD147, CD151, carcinoembryonic antigen (CEA), CD66a, CD326 (also known as epithelial cell adhesion molecule, EpCAM), CD49b, heat shock protein 60 (Hsp60), copine-3 (CPNE3), folate receptor alpha (FRa), CD24, vascular endothelial growth factor (VEGF), prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), epidermal growth factor receptor (EGFR), Glypican-3 (GPC-3), Glypican-1 (GPC-1), CD63, CD9, programmed death ligand 1 (PD-L1), leucine-rich alpha-2- glycoprotein 1 (LRG1), Epstein-Barr virus latent membrane protein 1 (LMP1), Epstein-Barr virus BamHI- A rightward frame 1 (BARF1), matrix metalloproteinase 9 (MMP9), ceruloplasmin (CP), Dickkopf-related protein 4 (DKK4), carbonic anhydrase 9 (CAIX), tumor-associated calcium signal transducer 2 (TACSTD2), CD36, CD44, Claudin 4, LI cell adhesion molecule (LI CAM), CD24, A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), extracellular matrix metalloproteinase inducer (EMMPRIN), transforming growth factor P-1 (TGF -1), melanoma antigen 3 / 6 (MAGE-3 / 6), CD37, CD53, CD81, CD47, CD26, CD196, CD274, CD133, and / or Ephrin A2), are conjugated to oligonucleotides. A connector oligonucleotide and a ligase are provided that result in ligation of the first and second oligonucleotides to each other if they are in proximity (as occurs when the binding agents (such as the antibodies) are bound to EV-associated target molecules in close proximity, or the EV- associated target molecule and an EV-specific marker). The oligonucleotides may include tags and / or barcodes as discussed above and as described elsewhere herein. The tags may have any ofAttorney Docket No.: GH0256WO the features described elsewhere herein with respect to tags. The ligation product can be a substrate for amplification. The ligation product can be detected (e.g., by sequencing or PCR, which may follow an amplification step), thus indicating co-localization of the two or more EV marker binding molecules. Where the presence, absence, or level of a plurality of target EV-associated target molecules is being detected, the assay may be multiplexed.H. Captured set; target regions

[0283] In some embodiments, nucleic acids captured or enriched using a method described herein comprise captured DNA, such as one or more captured sets of DNA. In some embodiments, the captured DNA comprise target regions that are differentially methylated in different immune cell types. In some embodiments, the immune cell types comprise rare or closely related immune cell types, such as activated and naive lymphocytes or myeloid cells at different stages of differentiation.

[0284] In some embodiments, a captured epigenetic target region set captured from a sample or first subsample comprises hypermethylation variable target regions. In some embodiments, the hypermethylation variable target regions are differentially or exclusively hypermethylated in one cell type or in one immune cell type, or in one immune cell type within a cluster. In some embodiments, the hypermethylation variable target regions are hypermethylated to an extent that is distinguishably higher or exclusively present in one cell type or one immune cell type or one immune cell type within a cluster. Such hypermethylation variable target regions may be hypermethylated in other cell types but not to the extent observed in the one cell type. In some embodiments, the hypermethylation variable target regions show lower methylation in healthy cfDNA than in at least one other tissue type.

[0285] In some embodiments, a captured epigenetic target region set captured from a sample or second subsample comprises hypomethylation variable target regions. In some embodiments, the hypomethylation variable target regions are exclusively hypomethylated in one cell type or in one immune cell type or in one immune cell type within a cluster. In some embodiments, the hypomethylation variable target regions are hypomethylated to an extent that is exclusively present in one cell type or one immune cell type or in one immune cell type within a cluster. Such hypomethylation variable target regions may be hypomethylated in other cell types but not to the extent observed in the one cell type. In some embodiments, the hypomethylation variable target regions show higher methylation in healthy cfDNA than in at least one other tissue type.Attorney Docket No.: GH0256WO

[0286] Without wishing to be bound by any particular theory, in an individual with cancer, proliferating or activated immune cells and / or cancer cells may shed more DNA into the bloodstream than immune cells in a healthy individual and / or healthy cells of the same tissue type, respectively. As such, the distribution of cell type and / or tissue of origin of cfDNA may change upon carcinogenesis. Thus, variations in hypermethylation and / or hypomethylation can be an indicator of disease. For example, an increase in the level of hypermethylation variable target regions and / or hypomethylation variable target regions in a subsample following a partitioning step can be an indicator of the presence (or recurrence, depending on the history of the subject) of cancer.

[0287] Exemplary hypermethylation variable target regions and hypomethylation variable target regions useful for distinguishing between various cell types, including but not limited to immune cell types, have been identified by analyzing DNA obtained from various cell types via whole gnome bisulfite sequencing, as described, e.g., in Scott, C.A., Duryea, J.D., MacKay, H. et al., “Identification of cell type-specific methylation signals in bulk whole genome bisulfite sequencing data,” Genome Biol 21, 156 (2020) (doi.org / 10.1186 / sl3059-020-02065-5). Wholegenome bisulfite sequencing data is available from the Blueprint consortium, available on the internet at dcc.blueprint-epigenome.eu.

[0288] In some embodiments, first and second captured target region sets comprise, respectively, DNA corresponding to a sequence-variable target region set and DNA corresponding to an epigenetic target region set, for example, as described in WO 2020 / 160414. The first and second captured sets may be combined to provide a combined captured set. The sequence-variable target region set and epigenetic target region set may have any of the features described for such sets in WO 2020 / 160414, which is incorporated by reference herein in its entirety. In some embodiments, the epigenetic target region set comprises a hypermethylation variable target region set. In some embodiments, the epigenetic target region set comprises a hypomethylation variable target region set. In some embodiments, the epigenetic target region set comprises CTCF binding regions. In some embodiments, the epigenetic target region set comprises fragmentation variable target regions. In some embodiments, the epigenetic target region set comprises transcriptional start sites. In some embodiments, the epigenetic target region set comprises regions that may show focal amplifications in cancer, e.g., one or more of AR, BRAF, CCND1, CCND2, CCNE1, CDK4, CDK6, EGFR, ERBB2, FGFR1, FGFR2, KIT, KRAS, MET, MYC, PDGFRA, PIK3CA, andAttorney Docket No.: GH0256WORAFI . For example, in some embodiments, the epigenetic target region set comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the foregoing targets.

[0289] In some embodiments, the sequence-variable target region set comprises a plurality of regions known to undergo somatic mutations in cancer. In some aspects, the sequence-variable target region set targets a plurality of different genes or genomic regions (“panel”) selected such that a determined proportion of subjects having a cancer exhibits a genetic variant or tumor marker in one or more different genes or genomic regions in the panel. The panel may be selected to limit a region for sequencing to a fixed number of base pairs. The panel may be selected to sequence a desired amount of DNA, e.g., by adjusting the affinity and / or amount of the probes as described elsewhere herein. The panel may be further selected to achieve a desired sequence read depth. The panel may be selected to achieve a desired sequence read depth or sequence read coverage for an amount of sequenced base pairs. The panel may be selected to achieve a theoretical sensitivity, a theoretical specificity, and / or a theoretical accuracy for detecting one or more genetic variants in a sample.

[0290] Probes for detecting the panel of regions can include those for detecting genomic regions of interest (hotspot regions). Information about chromatin structure can be taken into account in designing probes, and / or probes can be designed to maximize the likelihood that particular sites (e.g., KRAS codons 12 and 13) can be captured, and may be designed to optimize capture based on analysis of cfDNA coverage and fragment size variation impacted by nucleosome binding patterns and GC sequence composition. Regions used herein can also include non-hotspot regions optimized based on nucleosome positions and GC models.

[0291] Examples of listings of genomic locations of interest may be found in Table 3 and Table 4 of WO 2020 / 160414. In some embodiments, a sequence-variable target region set used in the methods of the present disclosure comprises at least a portion of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or 70 of the genes of Table 3 of WO 2020 / 160414. In some embodiments, a sequence-variable target region set used in the methods of the present disclosure comprises at least a portion of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, or 73 of the genes of Table 4 of WO 2020 / 160414. Additionally or alternatively, suitable target region sets are available from the literature. For example, Gale et al., PLoS One 13: e0194630 (2018), which is incorporatedAttorney Docket No.: GH0256WO herein by reference, describes a panel of 35 cancer-related gene targets that can be used as part or all of a sequence-variable target region set. These 35 targets are AKT1, ALK, BRAF, CCND1, CDK2A, CTNNB1, EGFR, ERBB2, ESRI, FGFR1, FGFR2, FGFR3, F0XL2, GATA3, GNA11, GNAQ, GNAS, FIRAS, IDH1, IDH2, KIT, KRAS, MED 12, MET, MYC, NFE2L2, NRAS, PDGFRA, PIK3CA, PPP2R1A, PTEN, RET, STK11, TP53, and U2AF1.

[0292] In some embodiments, the sequence-variable target region set comprises target regions from at least 10, 20, 30, or 35 cancer-related genes, such as the cancer-related genes listed above and in Tables 3 and 4 of WO 2020 / 160414.I. Sequencing

[0293] In general, sample proteins and / or nucleic acids, and / or nucleic acids generated (such as by an amplification step) from oligonucleotide labels disclosed herein (such as an oligonucleotide label illustrated in Fig. 1A), including nucleic acids flanked by adapters, with or without prior amplification can be subject to sequencing. Sequencing methods include, for example, Edman degradation based protein sequencing, mass spectrometry based protein sequencing, Sanger sequencing, high-throughput sequencing, pyrosequencing, sequencing-by-synthesis, singlemolecule sequencing, nanopore sequencing, semiconductor sequencing, sequencing-by-ligation, sequencing-by-hybridization, Digital Gene Expression (Helicos), Next generation sequencing (NGS), Single Molecule Sequencing by Synthesis (SMSS) (Helicos), massively-parallel sequencing, Clonal Single Molecule Array (Solexa), shotgun sequencing, Ion Torrent, Oxford Nanopore, Roche Genia, Maxim-Gilbert sequencing, primer walking, and sequencing using PacBio, SOLiD, Ion Torrent, or Nanopore platforms.

[0294] In some embodiments, sequencing comprises detecting and / or distinguishing unmodified and modified nucleobases. For example, single-molecule real-time (SMRT) sequencing facilitates direct detection of, e.g., 5-methylcytosine and 5-hydroxymethylcytosine as well as unmodified cytosine. See, e.g., Schatz., Nature Methods. 14(4): 347-348 (2017); and US 9,150,918. Sequencing reactions can be performed in a variety of sample processing units, which may multiple lanes, multiple channels, multiple wells, or other mean of processing multiple sample sets substantially simultaneously. Sample processing unit can also include multiple sample chambers to enable processing of multiple runs simultaneously.

[0295] The sequencing reactions can be performed on one or more forms of nucleic acids, such as those known to contain markers of cancer or of other disease. The sequencing reactions can alsoAttorney Docket No.: GH0256WO be performed on any nucleic acid fragments present in the sample. In some embodiments, sequence coverage of the genome may be less than 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9% or 100%. In some embodiments, the sequence reactions may provide for sequence coverage of at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of the genome. Sequence coverage can performed on at least 5, 10, 20, 70, 100, 200 or 500 different genes, or at most 5000, 2500, 1000, 500 or 100 different genes.

[0296] Simultaneous sequencing reactions may be performed using multiplex sequencing. In some cases, cell-free nucleic acids may be sequenced with at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, 100,000 sequencing reactions. In other cases cell-free nucleic acids may be sequenced with less than 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, 100,000 sequencing reactions. Sequencing reactions may be performed sequentially or simultaneously. Subsequent data analysis may be performed on all or part of the sequencing reactions. In some cases, data analysis may be performed on at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, 100,000 sequencing reactions. In other cases, data analysis may be performed on less than 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, 100,000 sequencing reactions. An exemplary read depth is 1000-50000 reads per locus (base).J. Sequencing Methods with Partitioning

[0297] As a variation on grouping sequencing reads of the same original molecule by molecular barcodes, a sample can be partitioned into aliquots as described in PCT / US2025 / 035226, incorporated by reference herein. Partitioning can be used either for individual samples or pooled samples, in which nucleic acids from different samples are distinguished by sample indexes. Partitioning preferably occurs before any amplification of original sample nucleic acid molecules so that amplicons of the same original molecule are not partitioned from each other. Partitioning reduces the number of instances of nucleic acid molecules having the same start and stop points in an individual aliquot relative to the sample before partitioning. Preferably the number of instances of nucleic acid molecules having the same start and stop points is reduced such that at least 75%, 80%, 90%, 95% or 99% of nucleic acid molecules in each aliquot have unique start and stop sequences.

[0298] The number of partitions depends on the characteristics of a population of nucleic acid molecules to be partitioned. These characteristics include the mean, median and mode of nucleicAttorney Docket No.: GH0256WO acid molecules having the same start and stop points, the maximum number of instances of nucleic acid molecules having the same start and stop points, and the overall distribution of instances of nucleic acid molecules having the same start and stop points.

[0299] For it to be statistically probable that an aliquot contains no instances of multiple nucleic acid molecules with the same start and stop points then the number of partitions should be equal to or greater (e.g., at least lx, 2x, 5 x or lOx) than the maximum number of instances of the same start and stop points in the sample before partition. Eight or sixteen partitions can sometimes be suitable.

[0300] With or without additional processing steps in separated partitions, the partitioned nucleic acid molecules can be labelled with partition indexes, such that nucleic acid molecules in the same aliquot receive the same partition index and nucleic acid molecules in at least some, and sometimes all of the different aliquots receive different partition indexes. Thus, linkage of sample molecules to partition indexes does not require random assortment of the partition indexes to the sample molecules. Partition indexes can be linked to sample molecules as primer components or by ligation, e.g., as a component of a further adapter. Preferably a partition index is included in one or both members of a pair of primers suitable for amplification of nucleic acid molecules in an aliquot. For example, such a primer pair can have 3’ regions complementary to adapter sequences flanking sample nucleic acid molecules, with one or both of the primers having a 5’ tail region including a partition index. If partition indexes are included in both members of a primer pair, the partition indexes can be the same or different from each other. After hybridization of such primers to adapter sequences, an amplification can conducted thereby covalently attaching partition indexes to sample nucleic acids.

[0301] An index is a short nucleic acid (e.g., less than 500, 100, 50, 20, 15, 10 or 5 nucleotides long), used to label nucleic acid molecules, for example to distinguish nucleic acids from different samples (a sample index), or nucleic acid molecules in different aliquots of the sample (partition indexes). The particular code stored by an index can be referred to as a designation of an index. Indexes are typically provided as sets of multiple different individual indexes for distinguishing samples or aliquots of a sample. That is, different samples receive different sample indexes from a set of sample indexes, and different aliquots receive different partition indexes.

[0302] In general, the distinction between a set of sample indexes and a set of partition indexes lies in the stages at which they added, the number of different indexes in the set, how the indexesAttorney Docket No.: GH0256WO are linked to samples nucleic acids, and the molecules they are used to distinguish rather than in indexes themselves. In principle, a set of sample indexes could be used as a set of partition indexes and vice versa. Preferably the code designations of a set of sample and partition indexes are mutually exclusive with one another.

[0303] After incorporation of partition indexes, further processing steps can be conducted on the aliquots separately or aliquots differentially labelled with partition indexes can be pooled and further processing steps performed on the pooled aliquots. Alternatively, the methods can be performed without use of partition indexes, in which case, all further processing steps are performed on separate aliquots so that it is known which sequencing reads originate from which aliquots. The methods can also be performed with some aliquots pooled and some kept separate from one another. The methods can also be performed with aliquots grouped in subpools, in which the aliquots within a subpool have different partition indexes from one another but aliquots in different subpools can have the same partition indexes as any of the other subpools. The different subpools are then kept separate from one another in subsequent processing whereas the aliquots within a subpooled are processed together. Sequencing reads can be traced back to the aliquot of origin based on a combination of the partition index present in a sequencing read and knowledge of the subpool from which it originated.

[0304] Further processing steps can include further amplification, affinity-enrichment for DNA molecules from selected genomic regions, sequencing and analysis of sequence reads. When partition indexes are used, sequencing is preferably performed after pooling of aliquots into a single vessel. Thus, nucleic acid molecules from the previously separate aliquots and different samples are sequenced together. When partition indexes are not used, sequencing is preferably performed keeping nucleic acid molecules from the different aliquots separate.

[0305] Sequencing reads from a sample are grouped to their molecule of origin by aliquot of origin determined by partition index or otherwise as described above, and a measure of sequence identity or similarity between sequencing reads. This measure can be start and stop points, which can be determined, for example, after alignment of sequencing reads with a reference sequence, length of sequencing reads, or minimum sequence similarity between reads (e.g., at least 95 or 99% identity after maximal alignment). If samples are pooled, sequence reads can be traced to a sample of origin from a sample index in the sequencing read. Ian information, when determined, can also be used in grouping sequencing reads. Grouping of sequencing reads by molecule of origin permitsAttorney Docket No.: GH0256WO distinction of genuine genetic or epigenetic variation from amplification and sequencing errors as further described below.

[0306] Methylation analysis can involve methylation-based separation of nucleic acid molecules. In some embodiments, methylation-based separation of nucleic acid molecules is performed by contacting the nucleic acid molecules with an agent that recognizes methylated DNA, such as 5- methylcytosine. In particular embodiments, the agent is a methyl binding reagent. In particular embodiments, the methyl binding reagent is a methyl binding domain (MBD) protein or an antibody. In some embodiments, the methyl binding reagent specifically recognizes 5- methylcytosine. For example, methylated fragments in a DNA sample can be separated via methylated DNA immunoprecipitation (MeDIP), or methylated fragments can be separated from unmethylated fragments using methyl binding domain proteins (e.g., MethylMinder Methylated DNA Enrichment Kit (ThermoFisher Scientific).

[0307] One application of partition methods is analysis of methylation state of nucleic acids. Methylation analysis can comprise subjecting parent nucleic acids or amplification products thereof to a procedure that affects a first nucleobase in the nucleic acid differently from a second nucleobase, for example wherein the first nucleobase is a modified or unmodified nucleobase, the second nucleobase is a modified or unmodified nucleobase different from the first nucleobase, and the first nucleobase and the second nucleobase have the same base pairing specificity. In some embodiments, the procedure that affects a first nucleobase of the nucleic acid differently from a second nucleobase of the nucleic acid is a methylation-sensitive conversion. In particular embodiments, the methylation-sensitive conversion is bisulfite conversion, oxidative bisulfite (Ox-BS) conversion, Tet-assisted bisulfite (TAB) conversion, APOBEC-coupled epigenetic (ACE) conversion, enzymatic methyl-seq (EM-seq) conversion, single-enzyme 5 -methyl cytosine sequencing (SEM-seq) conversion, or direct methylation sequencing (DM-seq).

[0308] Comparison of sequencing reads from treated and control groups indicates which cytosines were subject of modification. Splitting into groups for analysis of DNA modification is preferably performed after partitioning of samples or combined samples into aliquots so members of the same pairs of duplex strands are present in the same aliquot. Conversion also preferably precedes amplification. Conversion can occur before or after enrichment. If conversion occurs before enrichment, probes must be modified to hybridize with modified bases (e.g., U / T in place of C). Thus, a preferred order of steps is to attach sample indexes to different samples, pool the differentAttorney Docket No.: GH0256WO samples, partition the pooled samples, conversion of portions of the partitioned samples, amplification, enrichment and sequencing.

[0309] Methylation analysis can alternatively involve methylation-based separation of nucleic acid molecules. In some embodiments, methylation-based separation of nucleic acid molecules is performed by contacting the nucleic acid molecules with an agent that recognizes methylated DNA, such as 5-methylcytosine. In particular embodiments, the agent is a methyl binding reagent. In particular embodiments, the methyl binding reagent is a methyl binding domain (MBD) protein (e.g., see WO2018119452) or an antibody. In some embodiments, the methyl binding reagent specifically recognizes 5-methylcytosine. For example, methylated fragments in a DNA sample can be separated via methylated DNA immunoprecipitation (MeDIP), or methylated fragments can be separated from unmethylated fragments using methyl binding domain proteins (e.g., MethylMinder Methylated DNA Enrichment Kit (ThermoFisher Scientific). These types of methods separate DNA fragments having a high methyl C content from those with a low methyl C content before sequencing.

[0310] In one format, MBD separation is performed on individual samples, resulting in two portions for each sample, one having high methyl C content, the other lower methyl C content. The portions are then labelled with sample indexes. The portions are then pooled, high methyl content portions being pooled together, and low methyl content portions being pooled together. The two pools are then partitioned. Amplification and enrichment are performed in the separate partitions followed by attachment of partitions indexes. The partitions are then combined for sequencing. In another format, after ligation of sample indexes all portions are combined in the same pool instead of splitting into high and low methyl content pools. In another form, samples indexes are attached to samples before MBD separation. Thus, high and low methyl portions after MBD separation have the same sample index and are kept separate by pooling into two pools one with high methyl content, the other low methyl content. The two pools are separately portioned. The partitions are subject to amplification and enrichment followed by incorporation of partition indexes. The partitions are then combined for sequencing.

[0311] In some embodiments, sequencing of different aliquots is performed in different flow cells or different regions or lanes of the same flow cell. Different aliquots can be tracked using aliquotspecific partition indices (“Variation #1”) or tracked using partition indices and separate sequencing (“Variation #2”). In both variations, 96 samples, for example, are each ligated to aAttorney Docket No.: GH0256WO different sample index, and subsequently mixed and aliquoted into 96 wells. The particular numbers of samples and partitions are provided as an example. In Variation #1, each well receives a different partition index via PCR with labelled primers (i.e., the partition indices are aliquotspecific), and aliquots are subsequently pooled into a single pool prior to sequencing. The deconvolution of sequencing reads to original molecules is performed using the partition index, start / stop positions, and (for sample demultiplexing) the sample index. In Variation #2, each column of wells receives the same partition index whereas partition indices vary across each row, such that partition indices are aliquot-specific only with respect to a subset of the aliquots (and not all aliquots). In this variation, each row of aliquots is pooled (i.e., the pooling is amongst aliquots differentially labelled with partition indices), and each subset pool is sequenced separately). For example, each of the eight subset pools can be loaded onto a separate lane of a flow cell comprising eight lanes (or loaded on different flow cells or different sequencing instruments). The deconvolution of sequencing reads to original molecules is performed using the partition index, the separate sequencing, start / stop positions, and (for sample demultiplexing) the sample index.

[0312] In some embodiment, the methods do not necessarily involve an initial step of sample mixing before partitioning. Mixing or pooling nucleic acids from different samples after initial processing steps advantageously allows different samples to be subjected to different processing steps (e.g. different enrichment reactions). For example, in one embodiment, each of 96 samples is partitioned into eight aliquots, i.e. one column of wells per sample. The particular numbers of samples and partitions are provided as an example. Partition indices are introduced via PCR, wherein four different partition indices are used, such that two aliquots of each sample receive the same partition index. Aliquots of the same sample that have been differentially labelled with partition indices are then pooled such that two subset pools are generated per sample, which in turn means that two enrichment reactions are performed per sample (the enrichment reactions are performed on the subset pool). The two different subset pools deriving from the same sample are sequenced separately (e.g. in different lanes), and subsequent deconvolution of sequencing reads to original molecules is performed using the partition index, the separate sequencing, and start / stop positions. The partition indices may not be sample-specific (the partition indices are the same across rows), so the method can use tagging with sample indices before sample multiplexing. Alternatively, partition indices can be sample-specific, e.g. each column of wells can receive a different set of four partition indices; in such a case the ligation of separate indices for sampleAttorney Docket No.: GH0256WO demultiplexing is not required. Demultiplexing by sample of origin is based on the sample index or the sample-specific partition indexIII. Additional features of certain disclosed methodsA. Samples

[0313] A sample can be any biological sample isolated from a subject. A sample can be a bodily sample. Samples can include body tissues or fluids, such as known or suspected solid tumors, whole blood, platelets, serum, plasma, stool, red blood cells, white blood cells or leucocytes, endothelial cells, tissue biopsies, cerebrospinal fluid synovial fluid, lymphatic fluid, ascites fluid, interstitial or extracellular fluid, the fluid in spaces between cells, gingival crevicular fluid, bone marrow, pleural effusions, pleura fluid, cerebrospinal fluid, saliva, mucous, sputum, semen, sweat, and urine. Samples are preferably body fluids, particularly blood and fractions thereof, cerebrospinal fluid, pleura fluid, saliva, sputum, or urine. A sample can be in the form originally isolated from a subject or can have been subjected to further processing to remove or add components, such as cells, or enrich for one component relative to another. Thus, a preferred body fluid for analysis is plasma or serum, which may optionally contain cell-free nucleic acids.

[0314] In some embodiments, the sample comprises one or more target proteins (such as a first, second, third, fourth, and / or fifth target protein, or more target proteins), such as one or more target proteins comprising one or more PTMs. In some embodiments, a population of nucleic acids is obtained from a serum, plasma or blood sample from a subject suspected of having neoplasia, a tumor, precancer, or cancer or previously diagnosed with neoplasia, a tumor, precancer, or cancer. The population includes nucleic acids having varying levels of sequence variation, epigenetic variation, and / or post-replication or transcriptional modifications. Post-replication modifications include modifications of cytosine, particularly at the 5-position of the nucleobase, e.g., 5- methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5 -carboxyl cytosine.

[0315] A sample can be isolated or obtained from a subject and transported to a site of sample analysis. The sample may be preserved and shipped at a desirable temperature, e.g., room temperature, 4°C, -20°C, and / or -80°C. A sample can be isolated or obtained from a subject at the site of the sample analysis. The subject can be a human, a mammal, an animal, a companion animal, a service animal, or a pet. The subject may have a cancer, precancer, infection, transplant rejection, or other disease or disorder related to changes in the immune system. The subject mayAttorney Docket No.: GH0256WO not have cancer or a detectable cancer symptom. The subject may have been treated with one or more cancer therapy, e.g., any one or more of chemotherapies, antibodies, vaccines or biologies. The subject may be in remission. The subject may or may not be diagnosed of being susceptible to cancer or any cancer-associated genetic mutations / disorders.

[0316] In particular examples, the sample is a blood sample. In some embodiments, the blood sample is a whole blood sample, a plasma sample, a buffy coat sample, a leukapheresis sample, or a PBMC sample. In some embodiments, the sample comprises plasma obtained from a blood sample. In certain embodiments, the sample comprises serum. In certain embodiments, the sample is a tissue sample. In particular embodiments, the tissue sample is a biopsy, a fine needle aspirate, or a formalin-fixed paraffin-embedded tissue sample. In some embodiments, the sample comprises plasma. The volume of plasma obtained can depend on the desired read depth for sequenced regions. Exemplary volumes are 0.4-40 ml, 5-20 ml, 10-20 ml. For examples, the volume can be 0.5 mL, 1 mL, 5 mL 10 mL, 20 mL, 30 mL, or 40 mb. A volume of sampled plasma may be 5 to 20 mL. In some embodiments, the sample volume is 3-5 mL of plasma, such as 4 mL of plasma, per 10 mL whole blood.

[0317] In some embodiments, the sample comprises whole blood. Exemplary volumes of sampled whole blood are 0.4-40 mL, 5-20 mL, 10-20 mL, 1-6 mL, 1-3 mL, and 3-5 mL. For example, the volume can be 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 20 mL, 30 mL, or 40 mL. A volume of sampled whole blood may be 5 to 20 mL. In some embodiments, the sample volume is 1-5 mL of whole blood, such as 2.5 mL of whole blood.

[0318] In some embodiments, the sample comprises buffy coat separated from whole blood. Exemplary volumes of sampled buffy coat are 0.1-20 mL, 1-10 mL, 1-5 mL, 0.2-0.6 mL, and 0.3- 0.5 mL. For example, the volume can be 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL 10 mL, or 20 mL. A volume of sampled buffy coat may be 1 to 10 mL. In some embodiments, the sample volume is 0.1-0.5 mL of buffy coat, such as 0.3 mL of buffy coat, per 10 mL whole blood.

[0319] In some embodiments, the sample comprises PBMCs separated from whole blood. Exemplary volumes of sampled PBMCs are 0.1-20 mL, 1-10 mL, 1-5 mL, 0.2-0.6 mL, and 0.3- 0.5 mL. For example, the volume can be 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL 10 mL, or 20 mL. A volume of sampledAttorney Docket No.: GH0256WOPBMCs may be 1 to 10 mL. In some embodiments, the sample volume is 0.1-0.5 mL of PBMCs, such as 0.3 mL of PBMCs, per 10 mL whole blood.

[0320] In some embodiments, the sample comprises leukocytes separated from subject blood using leukapheresis. Exemplary volumes of sampled leukocytes from leukapheresis are 0.1-20 mL, 1-10 mL, 1-5 mL, 0.2-0.6 mL, and 0.3-0.5 mL. For example, the volume can be 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 10 mL, or 20 mL. A volume of sampled leukocytes from leukapheresis may be 1 to 10 mL. In some embodiments, the sample volume is 0.1-0.6 mL of leukocytes from leukapheresis, such as 0.4 mL of leukocytes, per 10 mL whole blood.

[0321] In some embodiments, extracellular vesicles comprising a plurality ofEV-associated target molecules are obtained from a serum, plasma or blood sample from a subject suspected of having neoplasia, a tumor, precancer, or cancer or previously diagnosed with neoplasia, a tumor, precancer, or cancer. In a particular embodiment, the sample comprises extracellular vesicles.

[0322] A sample can comprise EVs from different sources, e.g., EVs of the same subject, and EVs of different subjects. A sample can comprise EV-associated nucleic acids (e.g., DNA) carrying mutations. For example, a sample can comprise EV-associated DNA carrying germline mutations and / or somatic mutations. Germline mutations refer to mutations existing in germline DNA of a subject. Somatic mutations refer to mutations originating in somatic cells of a subject, e.g., cancer cells. A sample can comprise EV-associated DNA carrying cancer-associated mutations (e.g., cancer-associated somatic mutations). A sample can comprise an epigenetic variant (i.e., a chemical or protein modification), wherein the epigenetic variant associated with the presence of a genetic variant such as a cancer-associated mutation. In some embodiments, the sample comprises an epigenetic variant associated with the presence of a genetic variant, wherein the sample does not comprise the genetic variant.

[0323] Reference or control molecules can be added to or spiked into a sample as a control or normalization standard. For example, a certain amount of DNA or RNA from a species other than the species of the subject from which the sample was obtained or synthetic nucleic acids comprising certain modifications may be added to the sample. In some embodiments, the reference or control molecules are distinguishable from the molecules originally present in the sample. In some embodiments, detected oligonucleotides and / or EV-associated DNA or RNA sequences are normalized to the reference or control molecules.Attorney Docket No.: GH0256WOB. Capture moieties

[0324] As discussed above, molecules, such as proteins and / or nucleic acids in a sample can be subject to a capture step, in which target proteins or molecules having target regions are captured and analyzed. Target capture can involve use of oligonucleotides labeled with a capture moiety, such as biotin, and a second moiety or binding partner that binds to the capture moiety, such as streptavidin. In some embodiments, a capture moiety and binding partner can have higher and lower capture yields for different sets of target regions, such as those of the sequence-variable target region set and the epigenetic target region set, respectively, as discussed elsewhere herein. Methods comprising capture moieties are further described in, for example, U.S. patent 9,850,523, issuing December 26, 2017, which is incorporated herein by reference.

[0325] Capture moieties include, without limitation, biotin, avidin, streptavidin, a nucleic acid comprising a particular nucleotide sequence, a hapten recognized by an antibody, and magnetically attractable particles. The extraction moiety can be a member of a binding pair, such as biotin / streptavidin or hapten / antibody. In some embodiments, a capture moiety that is attached to an analyte is captured by its binding pair which is attached to an isolatable moiety, such as a magnetically attractable particle or a large particle that can be sedimented through centrifugation. The capture moiety can be any type of molecule that allows affinity separation of nucleic acids bearing the capture moiety from nucleic acids lacking the capture moiety. Exemplary capture moieties are biotin which allows affinity separation by binding to streptavidin linked or linkable to a solid phase or an oligonucleotide, which allows affinity separation through binding to a complementary oligonucleotide linked or linkable to a solid phase.C. Applications

[0326] The methods disclosed herein can facilitate analyzing a plurality of extracellular vesicle (EV)-associated target molecules in a sample. This information has utility in a wide range of contexts, including determining the detection of EV-associated target molecules, such as mutations in EV-associated nucleic acids (such as DNA), and in determining the presence or absence of a cancer in a subject.

[0327] The methods presented herein may be used as part of any method that benefits from obtaining an accurate EV-associated target molecule profile. This is because the methods disclosed herein allow for analyzing EV-associated target molecules in a sample using sequencing and / or amplification of oligonucleotides as disclosed herein, and / or EV-associatedAttorney Docket No.: GH0256WO nucleic acids. One useful exemplary application of the methods of the disclosure is using the resulting sequencing data in diagnosing and prognosing cancer or other genetic diseases or conditions, e.g., determining the presence or absence of a cancer in a subject.

[0328] Hence, in some embodiments, methods described herein comprise identifying or predicting the presence or absence of proteins or nucleic acids produced by a tumor (or neoplastic cells, or cancer cells), determining the likelihood that a test subject has a tumor or cancer, and / or characterizing a tumor, neoplastic cells or cancer as described herein.1. Cancer and other diseases; Cell Type Quantification

[0329] The present methods can be used to diagnose the presence of a condition, e.g., cancer or precancer, in a subject, to characterize a condition (such as to determine a cancer stage or heterogeneity of a cancer), to monitor a subject’s response to receiving a treatment for a condition (such as a response to a chemotherapeutic or immunotherapeutic), assess prognosis of a subject (such as to predict a survival outcome in a subject having a cancer), to determine a subject’s risk of developing a condition, to predict a subsequent course of a condition in a subject, to determine metastasis or recurrence of a cancer in a subject (or a risk of cancer metastasis or recurrence), and / or to monitor a subject’s health as part of a preventative health monitoring program (such as to determine whether and / or when a subject is in need of further diagnostic screening). The present disclosure can also be useful in determining the efficacy of a particular treatment option. Successful treatment options may increase the amount of target proteins, the number and / or type of PTMs on one or more of the target proteins, copy number variation, rare mutations, and / or cancer-related epigenetic signatures (such as hypermethylated regions or hypomethylated regions) detected in, e.g., a sample from a subject, such as detected in a subject's blood (such as in DNA isolated from a buffy coat sample or any other sample comprising cells, such as in a blood sample (e.g., a whole blood sample, a leukapheresis sample, or a PBMC sample) from the subject) if the treatment is successful as more cancer cells may die and shed DNA, or, e.g., if a successful treatment results in an increase or decrease in the quantity of a specific protein in the blood and an unsuccessful treatment results in no change. In other examples, this may not occur. In another example, certain treatment options may be correlated with profiles (e g., of target proteins and / or genetic profiles) of cancers over time. This correlation may be useful in selecting a therapy.

[0330] Additionally, if a cancer is observed to be in remission after treatment, the present methods can be used to monitor the likelihood of residual disease or the likelihood of recurrence of disease.Attorney Docket No.: GH0256WO

[0331] In some embodiments, the present methods are used for screening for a cancer, or in a method for screening cancer. For example, the sample can be a sample from a subject who has not been previously diagnosed with cancer. In some embodiments, the subject may or may not have cancer. In some embodiments, the subject may or may not have an early-stage cancer. In some embodiments, the subject has one or more risk factors for cancer, such as tobacco use (e.g., smoking), being overweight or obese, having a high body mass index (BMI), being of advanced age, poor nutrition, high alcohol consumption, or a family history of cancer. In some embodiments, the methods disclosed herein are used for screening for a cancer such as a metastasis, or in a method of detecting the presence or absence of a metastasis. In some embodiments, one or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more samples are collected from a subject as described herein, such as before and / or after the subject is diagnosed with a cancer.

[0332] In some embodiments, the subject has used tobacco, e.g., for at least 1, 5, 10, or 15 years. In some embodiments, the subject has a high BMI, e g., a BMI of 25 or greater, 26 or greater, 27 or greater, 28 or greater, 29 or greater, or 30 or greater. In some embodiments, the subject is at least 40, 45, 50, 55, 60, 65, 70, 75, or 80 years old. In some embodiments, the subject has poor nutrition, e.g., high consumption of one or more of red meat and / or processed meat, trans fat, saturated fat, and refined sugars, and / or low consumption of fruits and vegetables, complex carbohydrates, and / or unsaturated fats. High and low consumption can be defined, e.g., as exceeding or falling below, respectively, recommendations in Dietary Guidelines for Americans 2020-2025, available at dietaryguidelines.gov / sites / default / files / 2021-03 / Dietary_Guidelines_for_Americans-2020- 2025.pdf. In some embodiments, the subject has high alcohol consumption, e.g., at least three, four, or five drinks per day on average (where a drink is about one ounce or 30 mb of 80-proof hard liquor or the equivalent). In some embodiments, the subject has a family history of cancer, e.g., at least one, two, or three blood relatives were previously diagnosed with cancer. In some embodiments, the relatives are at least third-degree relatives (e.g., great-grandparent, great aunt or uncle, first cousin), at least second-degree relatives (e.g., grandparent, aunt or uncle, or halfsibling), or first-degree relatives (e g., parent or full sibling). Furthermore, in some embodiments, the one or more methods described in the present disclosure may be used to assist in the treatment of a type of cancer.

[0333] In some embodiments, the methods and systems disclosed herein may be used to identify customized or targeted therapies to treat a given disease or condition in patients based on theAttorney Docket No.: GH0256WO presence of one or more proteins of interest (i.e., one or more target proteins), the presence and / or absence of one or more PTMs on the one or more target proteins, and / or classification of a nucleic acid variant as being of somatic or germline origin. Typically, the disease under consideration is a type of cancer. Non-limiting examples of such cancers include biliary tract cancer, bladder cancer, transitional cell carcinoma, urothelial carcinoma, brain cancer, gliomas, astrocytomas, breast carcinoma, metaplastic carcinoma, cervical cancer, cervical squamous cell carcinoma, rectal cancer, colorectal carcinoma, colon cancer, hereditary nonpolyposis colorectal cancer, colorectal adenocarcinomas, gastrointestinal stromal tumors (GISTs), endometrial carcinoma, endometrial stromal sarcomas, esophageal cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, ocular melanoma, uveal melanoma, gallbladder carcinomas, gallbladder adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma, transitional cell carcinoma, urothelial carcinomas, Wilms tumor, leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), liver cancer, liver carcinoma, hepatoma, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, Lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, B-cell lymphomas, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, Mantle cell lymphoma, T cell lymphomas, non-Hodgkin lymphoma, precursor T- lymphoblastic lymphoma / leukemia, peripheral T cell lymphomas, multiple myeloma, nasopharyngeal carcinoma (NPC), neuroblastoma, oropharyngeal cancer, oral cavity squamous cell carcinomas, osteosarcoma, ovarian carcinoma, pancreatic cancer, pancreatic ductal adenocarcinoma, pseudopapillary neoplasms, acinar cell carcinomas. Prostate cancer, prostate adenocarcinoma, skin cancer, melanoma, malignant melanoma, cutaneous melanoma, small intestine carcinomas, stomach cancer, gastric carcinoma, gastrointestinal stromal tumor (GIST), uterine cancer, or uterine sarcoma. Type and / or stage of cancer can be detected from genetic variations including mutations, rare mutations, indels, rearrangements, copy number variations, transversions, translocations, recombinations, inversion, deletions, aneuploidy, partial aneuploidy, polyploidy, chromosomal instability, chromosomal structure alterations, gene fusions, chromosome fusions, gene truncations, gene amplification, gene duplications, chromosomal lesions, DNA lesions, abnormal changes in nucleic acid chemical modifications, abnormal changes in epigenetic patterns, and abnormal changes in nucleic acid 5 -methylcytosine.Attorney Docket No.: GH0256WO

[0334] Target protein and genetic data can also be used for characterizing a specific form of cancer. Cancers are often heterogeneous in both composition and staging. Genetic profile data may allow characterization of specific sub-types of cancer that may be important in the diagnosis or treatment of that specific sub-type. This information may also provide a subject or practitioner clues regarding the prognosis of a specific type of cancer and allow either a subject or practitioner to adapt treatment options in accord with the progress of the disease. Some cancers can progress to become more aggressive and genetically unstable. Other cancers may remain benign, inactive or dormant. The system and methods of this disclosure may be useful in determining disease progression.

[0335] Further, the methods of the disclosure may be used to characterize the heterogeneity of an abnormal condition in a subject. Such methods can include, e.g., generating a genetic profile of extracellular molecules and polynucleotides derived from the subject, wherein the genetic profile comprises a plurality of data resulting from copy number variation and rare mutation analyses. In some embodiments, an abnormal condition is cancer. In some embodiments, the abnormal condition may be one resulting in a heterogeneous genomic population. In the example of cancer, some tumors are known to comprise tumor cells in different stages of the cancer. In other examples, heterogeneity may comprise multiple foci of disease. Again, in the example of cancer, there may be multiple tumor foci, perhaps where one or more foci are the result of metastases that have spread from a primary site.

[0336] The present methods can be used to generate or profile, fingerprint or set of data that is a summation of target protein and genetic information derived from different cells in a heterogeneous disease. This set of data may comprise protein levels (e.g., of one or more target proteins), amounts and / or types of PTMs on one or more target proteins, copy number variation, epigenetic variation, and mutation analyses alone or in combination.

[0337] The present methods can be used to diagnose, prognose, monitor or observe cancers, precancers, or other diseases. In some embodiments, the methods herein do not involve the diagnosing, prognosing or monitoring a fetus and as such are not directed to non-invasive prenatal testing. In other embodiments, these methodologies may be employed in a pregnant subject to diagnose, prognose, monitor or observe cancers or other diseases in an unborn subject whose DNA and other polynucleotides may co-circulate with maternal molecules.Attorney Docket No.: GH0256WO

[0338] Non-limiting examples of other genetic-based diseases, disorders, or conditions that are optionally evaluated using the methods and systems disclosed herein include achondroplasia, alpha- 1 antitrypsin deficiency, antiphospholipid syndrome, autism, autosomal dominant polycystic kidney disease, Charcot-Marie-Tooth (CMT), cri du chat, Crohn's disease, cystic fibrosis, Dercum disease, down syndrome, Duane syndrome, Duchenne muscular dystrophy, Factor V Leiden thrombophilia, familial hypercholesterolemia, familial Mediterranean fever, fragile X syndrome, Gaucher disease, hemochromatosis, hemophilia, holoprosencephaly, Huntington's disease, Klinefelter syndrome, Marfan syndrome, myotonic dystrophy, neurofibromatosis, Noonan syndrome, osteogenesis imperfecta, Parkinson's disease, phenylketonuria, Poland anomaly, porphyria, progeria, retinitis pigmentosa, severe combined immunodeficiency (SCID), sickle cell disease, spinal muscular atrophy, Tay-Sachs, thalassemia, trimethylaminuria, Turner syndrome, velocardiofacial syndrome, WAGR syndrome, Wilson disease, or the like.

[0339] In some embodiments, a method described herein comprises detecting a presence or absence of one or more target proteins, and / or detecting a presence or absence of one or more PTMs on one or more target proteins, originating or derived from a tumor cell at a preselected timepoint following a previous cancer treatment of a subject previously diagnosed with cancer. DNA originating or derived from the tumor cell may also be detected. The method may further comprise determining a cancer recurrence score that is indicative of the presence or absence of the target protein and, where applicable, DNA originating or derived from the tumor cell for the subject. In some embodiments, the sample is obtained from a subject who was previously diagnosed with a cancer and received one or more previous cancer treatments. In some embodiments, the sample is obtained at one or more preselected time points following the one or more previous cancer treatments.

[0340] Where a cancer recurrence score is determined, it may further be used to determine a cancer recurrence status. The cancer recurrence status may be at risk for cancer recurrence, e.g., when the cancer recurrence score is above a predetermined threshold. The cancer recurrence status may be at low or lower risk for cancer recurrence, e.g., when the cancer recurrence score is above a predetermined threshold. In particular embodiments, a cancer recurrence score equal to the predetermined threshold may result in a cancer recurrence status of either at risk for cancer recurrence or at low or lower risk for cancer recurrence.Attorney Docket No.: GH0256WO

[0341] In some embodiments, a cancer recurrence score is compared with a predetermined cancer recurrence threshold, and the subject is classified as a candidate for a subsequent cancer treatment when the cancer recurrence score is above the cancer recurrence threshold or not a candidate for therapy when the cancer recurrence score is below the cancer recurrence threshold. In particular embodiments, a cancer recurrence score equal to the cancer recurrence threshold may result in classification as either a candidate for a subsequent cancer treatment or not a candidate for therapy.

[0342] The methods discussed above may further comprise any compatible feature or features set forth elsewhere herein, including in the section regarding methods of determining a risk of cancer recurrence in a subject and / or classifying a subject as being a candidate for a subsequent cancer treatment.2. Methods of determining a risk of cancer recurrence in a subject and / or classifying a subject as being a candidate for a subsequent cancer treatment

[0343] In some embodiments, a method provided herein is a method of determining a risk of cancer recurrence in a subject. In some embodiments, a method provided herein is a method of classifying a subject as being a candidate for a subsequent cancer treatment. In some embodiments, a method provided herein is or comprises a method of detecting the presence or absence of a metastasis in a subject.

[0344] Any of such methods may comprise collecting a sample (such as EVs originating or derived from a tumor) from the subject diagnosed with the cancer at one or more preselected timepoints following one or more previous cancer treatments to the subject. The subject may be any of the subjects described herein. The sample may comprise proteins from dead or dying cells. The sample may comprise DNA, e.g., cfDNA. The DNA may be obtained from a tissue sample or a blood sample (e.g., a whole blood sample, a buffy coat sample, a leukapheresis sample, or a PBMC sample).

[0345] Any of such methods may comprise contacting the sample or a subsample thereof with at least one binding molecule and detecting the presence or level of one or more target proteins, and / or detecting the presence or level of one or more PTMs on the one or more target proteins, according to any of the embodiments as described herein. The methods may further comprise capturing a plurality of sets of target regions from DNA from the subject, wherein the plurality of target region sets comprise a sequence-variable target region set, and / or an epigenetic target region set, whereby a captured set of DNA molecules is produced. The capturing step may be performedAttorney Docket No.: GH0256WO according to any of the embodiments described elsewhere herein. Any of such methods may comprise sequencing the captured DNA molecules, whereby a set of sequence information is produced. The captured DNA molecules of a sequence-variable target region set may be sequenced to a greater depth of sequencing than the captured DNA molecules of the epigenetic target region set. Any of such methods may comprise detecting a presence or absence of DNA originating or derived from a tumor cell at a preselected timepoint using the set of sequence information. The detection of the presence or absence of DNA originating or derived from a tumor cell may be performed according to any of the embodiments thereof described elsewhere herein.

[0346] In any of such methods, the previous cancer treatment may comprise surgery, administration of a therapeutic composition, and / or chemotherapy.

[0347] Methods of determining a risk of cancer recurrence in a subject may comprise determining a cancer recurrence score that is indicative of the presence or absence, or amount, of one or more target proteins and / or one or more nucleic acids originating or derived from the tumor cell for the subject, and / or is indicative of the presence or amount of one or more PTMs on the one or more target proteins originating or derived from the tumor cell for the subject. The cancer recurrence score may further be used to determine a cancer recurrence status. The cancer recurrence status may be at risk for cancer recurrence, e.g., when the cancer recurrence score is above a predetermined threshold. The cancer recurrence status may be at low or lower risk for cancer recurrence, e.g., when the cancer recurrence score is above a predetermined threshold. In particular embodiments, a cancer recurrence score equal to the predetermined threshold may result in a cancer recurrence status of either at risk for cancer recurrence or at low or lower risk for cancer recurrence.

[0348] Methods of detecting the presence or absence of metastasis in a subject may comprise comparing the presence or level of a tissue-specific cell material to the presence or level of the tissue-specific cell material obtained from the subject at a different time, a reference level of the tissue-specific cell material, or to a comparator cell material. Methods herein may comprise additional steps to determine whether a metastasis is present.

[0349] Methods of classifying a subject as being a candidate for a subsequent cancer treatment may comprise comparing the cancer recurrence score of the subject with a predetermined cancer recurrence threshold, thereby classifying the subject as a candidate for the subsequent cancer treatment when the cancer recurrence score is above the cancer recurrence threshold or not aAttorney Docket No.: GH0256WO candidate for therapy when the cancer recurrence score is below the cancer recurrence threshold. In particular embodiments, a cancer recurrence score equal to the cancer recurrence threshold may result in classification as either a candidate for a subsequent cancer treatment or not a candidate for therapy. In some embodiments, the subsequent cancer treatment comprises chemotherapy or administration of a therapeutic composition.

[0350] Any of such methods may comprise determining a disease-free survival (DFS) period for the subject based on the cancer recurrence score; for example, the DFS period may be 1 year, 2 years, 3, years, 4 years, 5 years, or 10 years.

[0351] In some embodiments, the set of sequence information comprises sequence-variable target region sequences and determining the cancer recurrence score may comprise determining at least a first subscore indicative of the levels of particular immune cell types, SNVs, insertions / deletions, CNVs and / or fusions present in sequence-variable target region sequences.

[0352] In some embodiments, a number of mutations in the sequence-variable target regions chosen from 1, 2, 3, 4, or 5 is sufficient for the first subscore to result in a cancer recurrence score classified as positive for cancer recurrence. In some embodiments, the number of mutations is chosen from 1 , 2, or 3.

[0353] In any embodiment where a cancer recurrence score is classified as positive for cancer recurrence, the cancer recurrence status of the subject may be at risk for cancer recurrence and / or the subject may be classified as a candidate for a subsequent cancer treatment.

[0354] In some embodiments, the cancer is any one of the types of cancer described elsewhere herein, e.g., colorectal cancer.3. Methods of monitoring a cancer in a subject over time; sample collection at two or more time points

[0355] In some embodiments, the present methods can be used to monitor one or more aspects of a condition in a subject over time, such as a subject’s response to receiving a treatment for a condition (such as a response to a chemotherapeutic or immunotherapeutic), the severity of the condition (such as a cancer stage) in the subject, a recurrence of the condition (such as a cancer), and / or the subject’s risk of developing the condition (such as a cancer) and / or to monitor a subject’s health as part of a preventative health monitoring program (such as to determine whether and / or when a subject is in need of further diagnostic screening). In some embodiments, monitoringAttorney Docket No.: GH0256WO comprises analysis of at least two samples collected from a subject at at least two different time points as described herein.

[0356] The methods according to the present disclosure can also be useful in predicting a subject’s response to a particular treatment option. Successful treatment options may result in an increase or decrease in the quantity of one or more target proteins and / or the quantity and / or type of one or more PTMs on the one or more target proteins (e.g., in the blood), and an unsuccessful treatment may result in no change. In other examples, this may not occur. In another example, certain treatment options may be correlated with profdes (e.g., of target proteins and / or genetic profiles) of cancers over time. This correlation may be useful in selecting a therapy for a subject.

[0357] The disclosed methods can include evaluating (such as quantifying) and / or interpreting a protein or proteins present in one or more samples comprising cells or a blood sample (e.g., a buffy coat sample, a whole blood sample, a leukapheresis sample, or a PBMC sample), collected from a subject at one or more timepoints in comparison to a selected baseline value or reference standard (or a selected set of baseline values or reference standards). A baseline value or reference standard may be a quantity of the one or more target proteins, and / or a quantity or type of one or more PTMs on the one or more target proteins, measured in one or more samples (such as an average quantity or range of quantities of the protein or proteins present in at least two samples) collected from the subj ect at one or more time points, such as prior to receiving a treatment, prior to diagnosis of a condition (such as a cancer), or as part of a preventative health monitoring program. A baseline value or reference standard may be a quantity of the protein or proteins measured in one or more samples (such as an average quantity or range of quantities of the protein or proteins present in at least two samples) collected at one or more timepoints from one or more subjects that do not have the condition (such as a healthy subject that does not have a cancer), one or more subjects that responded favorably to the treatment, or one or more subjects that have not received the treatment. In certain embodiments, the baseline value or reference standard utilized is a standard or profile derived from a single reference subject. In other embodiments, the baseline value or reference standard utilized is a standard or profile derived from averaged data from multiple reference subjects. The reference standard, in various embodiments, can be a single value, a mean, an average, a numerical mean or range of numerical means, a numerical pattern, or a graphical pattern created from the cell type quantity data derived from a single reference subject or from multiple reference subjects. Selection of the particular baseline values or reference standards, or selectionAttomey Docket No.: GH0256WO of the one or more reference subjects, depends upon the use to which the methods described herein are to be put by, for example, a research scientist or a clinician (such as a physician).

[0358] In some embodiments, one or more samples (such as a sample comprising cells or a blood sample (e.g., a buffy coat sample, a whole blood sample, a leukapheresis sample, or a PBMC sample) may be collected from a subject at two or more timepoints, to assess changes in a protein or proteins (such as changes in quantities of the protein or proteins, or changes in one or more modifications (such as one or more post-translational modifications) of the protein or proteins) between the two or more timepoints. In some embodiments, a sample collected at a first time point is a tissue sample or a blood sample, and a sample collected at a subsequent time point (such as a second time point) is a blood sample. In some embodiments, a sample collected at a first time point is a tissue sample and a sample collected at a subsequent time point (such as a second time point) is a blood sample. By monitoring a protein or proteins and identifying differences between the protein or proteins in samples collected from a subject at two or more timepoints, the present methods can be used, for example, to determine the presence or absence of a condition (such as a cancer), a response of the subject to a treatment, one or more characteristic of a condition (such as a cancer stage) in the subject, recurrence of a condition (such as a cancer), and / or a subject’s risk of developing a condition (such as a cancer). Thus, in some embodiments, methods are provided wherein quantities of a protein or proteins present in at least one sample (such as at least one whole blood sample, buffy coat sample, leukapheresis sample, or PBMC sample) collected from a subject at one or more timepoints (such as prior to receiving a treatment) are compared to quantities of the protein or proteins present in at least one sample collected from the subject at one or more different time points (such as after receiving the treatment). The disclosed methods can allow for patientspecific monitoring, such that, for example, differences in protein quantities and / or protein modifications between samples collected from the subject at different timepoints may indicate changes (such as presence or absence of a condition, response to a treatment, a prognosis, or the like) that are significant with respect to the subject but may yet fall within a normal range of a general healthy population.

[0359] As disclosed herein, methods are provided for monitoring one or more aspects of a condition in a subject over time, such as but not limited to, a subject’s response to receiving a treatment for a condition (such as a response to a chemotherapeutic or immunotherapeutic). In certain embodiments, one or more samples is collected from the subject at at least 1-10, at least 1-Attorney Docket No.: GH0256WO5, at least 2-5, or at least 1, at least 2, least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 time points prior to the subj ect receiving the treatment. In certain embodiments, one or more samples is collected from the subject at at least 1-10, at least 1-5, at least 2-5, or at least 1, at least 2, least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 time points after the subject has received the treatment. Sample collection from a subject can be ongoing during and / or after treatment to monitor the subject’s response to the treatment.

[0360] In some embodiments, samples are not collected from a subject prior to diagnosis of a condition (such as a cancer) or prior to receiving a treatment. In such embodiments, wherein the response of a subject to a treatment, or the course or stage of a condition (such as a cancer) in the subject is being monitored over time, cell types are compared between samples taken at at least 2- 10, at least 2-5, at least 3-6, or at least 2, such as at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 time points collected after the subject has been diagnosed and / or after the subject has received the treatment. Sample collection from a subject can be ongoing during and / or after treatment to monitor the subject’s response to the treatment.

[0361] In some embodiments of the disclosed methods, one or more samples, such as a sample comprising cells or a blood sample (such as one or more whole blood, buffy coat, leukapheresis, or PBMC samples) is collected from a subject at least once per year, such as about 1-12 times or about 2-6 times, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times per year. In other embodiments, one or more samples is collected from the subject less than once per year, such as about once every 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months. In some embodiments, one or more samples is collected from the subject about once every 1-5 years or about once every 1-2 years, such as about every 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 years.

[0362] In other embodiments of the disclosed methods, one or more samples, such one or more samples comprising cells or one or more blood samples, e.g., one or more buffy coat samples, whole blood samples, leukapheresis samples, or PBMC samples, are collected from a subject at least once per week, such as on 1-4 days, 1-2 days, or on 1, 2, 3, 4, 5, 6, or 7 days per week. In certain embodiments, one or more samples is collected from the subject at least once per month, such as 1-15 times, 1-10 times, 2-5 times, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 times per month. In other embodiments, one or more samples is collected from the subject every month,Attorney Docket No.: GH0256WO every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, or every 12 months. In some embodiments, one or more samples is collected from the subject at least once per day, such as 1, 2, 3, 4, 5, or 6 times per day. Selection of the one or more sample collection timepoints (e.g., the frequency of sample collection), or of the number of samples to be collected at each timepoint, depends upon the use to which the methods described herein are to be put by, for example, a research scientist or a clinician (such as a physician).

[0363] In certain embodiments, the customized therapies described herein are typically administered parenterally (e.g., intravenously or subcutaneously). Pharmaceutical compositions containing an immunotherapeutic agent are typically administered intravenously. Certain therapeutic agents are administered orally. However, customized therapies (e.g., immunotherapeutic agents, etc.) may also be administered by methods such as, for example, buccal, sublingual, rectal, vaginal, intraurethral, topical, intraocular, intranasal, and / or intraauricular, which administration may include tablets, capsules, granules, aqueous suspensions, gels, sprays, suppositories, salves, ointments, or the like.

[0364] Therapeutic options for treating specific genetic-based diseases, disorders, or conditions, other than cancer, are generally well-known to those of ordinary skill in the art and will be apparent given the particular disease, disorder, or condition under consideration.4. Therapies and Related Administration

[0365] In certain embodiments, the methods disclosed herein relate to identifying and administering therapies, such as customized therapies, to patients or subjects. In some embodiments, determination of the levels of particular target proteins, and / or the level and / or type of one or more PTMs on the target protein(s) facilitates selection of appropriate treatment.

[0366] In some embodiments, the patient or subject has a given disease, disorder or condition (e.g., any of the cancers or other conditions described elsewhere herein). Essentially any cancer therapy (e.g., surgical therapy, radiation therapy, chemotherapy, immunotherapy, and / or the like) may be included as part of these methods. In certain embodiments, the therapy administered to a subject comprises at least one chemotherapy drug. In some embodiments, the chemotherapy drug may comprise alkylating agents (for example, but not limited to, Chlorambucil, Cyclophosphamide, Cisplatin and Carboplatin), nitrosoureas (for example, but not limited to, Carmustine and Lomustine), anti-metabolites (for example, but not limited to, Fluorauracil, Methotrexate andAttorney Docket No.: GH0256WOFludarabine), plant alkaloids and natural products (for example, but not limited to, Vincristine, Paclitaxel and Topotecan), anti- tumor antibiotics (for example, but not limited to, Bleomycin, Doxorubicin and Mitoxantrone), hormonal agents (for example, but not limited to, Prednisone, Dexamethasone, Tamoxifen and Leuprolide) and biological response modifiers (for example, but not limited to, Herceptin and Avastin, Erbitux and Rituxan). In some embodiments, the chemotherapy administered to a subject may comprise FOLFOX or FOLFIRI. In certain embodiments, a therapy may be administered to a subject that comprises at least one PARP inhibitor. In certain embodiments, the PARP inhibitor may include OLAPARIB, TALAZOPARIB, RUCAPARIB, NIRAPARIB (trade name ZEJULA), among others. Typically, therapies include at least one immunotherapy (or an immunotherapeutic agent). Immunotherapy refers generally to methods of enhancing an immune response against a given cancer type. In certain embodiments, immunotherapy refers to methods of enhancing a T cell response against a tumor or cancer.

[0367] In some embodiments, therapy is customized based on the status of a nucleic acid variant as being of somatic or germline origin. In some embodiments, essentially any cancer therapy (e.g., surgical therapy, radiation therapy, chemotherapy, immunotherapy and / or the like) may be included as part of these methods. Customized therapies include at least one immunotherapy (or an immunotherapeutic agent). Immunotherapy refers generally to methods of enhancing an immune response against a given cancer type. In certain embodiments, immunotherapy refers to methods of enhancing a T cell response against a tumor or cancer.

[0368] In some embodiments, the immunotherapy or immunotherapeutic agent targets an immune checkpoint molecule. Certain tumors are able to evade the immune system by co-opting an immune checkpoint pathway. Thus, targeting immune checkpoints has emerged as an effective approach for countering a tumor’s ability to evade the immune system and activating anti-tumor immunity against certain cancers. Pardoll, Nature Reviews Cancer, 2012, 12:252-264.

[0369] In some embodiments the treatment comprises immunotherapies and / or immune checkpoint inhibitors (ICIS). Immunotherapies are treatments with one or more agents that act to stimulate the immune system so as to kill or at least to inhibit growth of cancer cells, and preferably to reduce further growth of the cancer, reduce the size of the cancer and / or eliminate the cancer. Some such agents bind to a target present on cancer cells; some bind to a target present on immune cells and not on cancer cells; some bind to a target present on both cancer cells and immune cells. Such agents include, but are not limited to, checkpoint inhibitors and / or antibodies. CheckpointAttorney Docket No.: GH0256WO inhibitors are inhibitors of pathways of the immune system that maintain self-tolerance and modulate the duration and amplitude of physiological immune responses in peripheral tissues to minimize collateral tissue damage (see, e.g., Pardoll, Nature Reviews Cancer 12, 252-264 (2012)). Exemplary agents include antibodies against any of PD-1, PD-2, PD-L1, PD-L2, CTLA-4, 0X40, B7.1, B7He, LAG3, CD137, KIR, CCR5, CD27, CD40, or CD47. Other exemplary agents include proinflammatory cytokines, such as IL-10, IL-6, and TNF-ot. Other exemplary agents are T-cells activated against a tumor, such as T-cells activated by expressing a chimeric antigen targeting a tumor antigen recognized by the T-cell. In some embodiments, anti-PD-1 or anti-PD-Ll therapies comprise pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), and cemiplimab (LIBTAYO®), atezolizumab (TECENTRIQ®), durvalumab (INFINZI®), and avelumab (BAVENCIO®). These therapies may be used to treat patients identified as having high microsatellite instability (MSI) status or high tumor mutational burden (TMB).

[0370] In certain embodiments, the immune checkpoint molecule is an inhibitory molecule that reduces a signal involved in the T cell response to antigen. For example, CTLA4 is expressed on T cells and plays a role in downregulating T cell activation by binding to CD80 (akaB7.1) or CD86 (aka B7.2) on antigen presenting cells. PD-1 is another inhibitory checkpoint molecule that is expressed on T cells. PD-1 limits the activity of T cells in peripheral tissues during an inflammatory response. In addition, the ligand for PD-1 (PD-L1 or PD-L2) is commonly upregulated on the surface of many different tumors, resulting in the downregulation of anti-tumor immune responses in the tumor microenvironment. In certain embodiments, the inhibitory immune checkpoint molecule is CTLA4 or PD-1. In other embodiments, the inhibitory immune checkpoint molecule is a ligand for PD-1, such as PD-L1 or PD-L2. In other embodiments, the inhibitory immune checkpoint molecule is a ligand for CTLA4, such as CD80 or CD86. In other embodiments, the inhibitory immune checkpoint molecule is lymphocyte activation gene 3 (LAG3), killer cell immunoglobulin like receptor (KIR), T cell membrane protein 3 (TIM3), galectin 9 (GAL9), or adenosine A2a receptor (A2aR).

[0371] Antagonists that target these immune checkpoint molecules can be used to enhance antigen-specific T cell responses against certain cancers. Accordingly, in certain embodiments, the immunotherapy or immunotherapeutic agent is an antagonist of an inhibitory immune checkpoint molecule. In certain embodiments, the inhibitory immune checkpoint molecule is PD-1. In certain embodiments, the inhibitory immune checkpoint molecule is PD-L1. In certain embodiments, theAttorney Docket No.: GH0256WO antagonist of the inhibitory immune checkpoint molecule is an antibody (e.g., a monoclonal antibody). In certain embodiments, the antibody or monoclonal antibody is an anti-CTLA4, anti- PD-1, anti-PD-Ll, or anti-PD-L2 antibody. In certain embodiments, the antibody is a monoclonal anti-PD-1 antibody. In some embodiments, the antibody is a monoclonal anti-PD-Ll antibody. In certain embodiments, the monoclonal antibody is a combination of an anti-CTLA4 antibody and an anti-PD-1 antibody, an anti-CTLA4 antibody and an anti-PD-Ll antibody, or an anti-PD-Ll antibody and an anti-PD-1 antibody. In certain embodiments, the anti-PD-1 antibody is one or more of pembrolizumab (Keytruda®) or nivolumab (Opdivo®). In certain embodiments, the anti- CTLA4 antibody is ipilimumab (Yervoy®). In certain embodiments, the anti-PD-Ll antibody is one or more of atezolizumab (Tecentriq®), avelumab (Bavencio®), or durvalumab (Imfinzi®).

[0372] In certain embodiments, the immunotherapy or immunotherapeutic agent is an antagonist (e.g. antibody) against CD80, CD86, LAG3, KIR, TIM3, GAL9, or A2aR. In other embodiments, the antagonist is a soluble version of the inhibitory immune checkpoint molecule, such as a soluble fusion protein comprising the extracellular domain of the inhibitory immune checkpoint molecule and an Fc domain of an antibody. In certain embodiments, the soluble fusion protein comprises the extracellular domain of CTLA4, PD-1, PD-L1, or PD-L2. In some embodiments, the soluble fusion protein comprises the extracellular domain of CD80, CD86, LAG3, KIR, TIM3, GAL9, or A2aR. In one embodiment, the soluble fusion protein comprises the extracellular domain of PD-L2 or LAG3.

[0373] In some embodiments, the therapies target mutated forms of the EGFR protein. Such therapies can include osimertinib (TAGRISSO®), erlotinib (TARCEVA®), and gefinitib (IRES SA®).

[0374] Therapies can include one or more of treatments for target therapies, including abemaciclib (VERZENIO®), abiraterone acetate (ZYTIGA®), acalabrutinib (CALQUENCE®), adagrasib (KRAZATI®), ado-trastuzumab emtansine (KADCYLA®), afatinib dimaleate (GILOTRIF®), alectinib (ALCENSA®), alemtuzumab (CAMPATH®), alitretinoin (PANRETIN®), alpelisib (PIQRAY®), amivantamab- vmjw (RYBREVANT®), anastrozole (ARIMIDEX®), apalutamide (ERLEADA®), asciminib hydrochloride (SCEMBLIX®), atezolizumab (TECENTRIQ®), avapritinib (AYVAKIT®), avelumab (BAVENCIO®), axicabtagene ciloleucel (YESCARTA®), axitinib (INLYTA®), belinostat (BELEODAQ®), belzutifan (WELIREG®), bevacizumab (AVASTIN®), bexarotene (TARGRETIN®),Attorney Docket No.: GH0256WO binimetinib (MEKTOVI®), blinatumomab (BLINCYTO®), bortezomib (VELCADE®), bosutinib (BOSULIF®), brentuximab vedotin (ADCETRIS®), brexucabtagene autoleucel (TECARTUS®), brigatinib (ALUNBRIG®), cabazitaxel (JEVTANA), cabozantinib-s-malate (CABOMETYX®), cabozantinib-s-malate (COMETRIQ®), capmatinib hydrochloride (TABRECTA®), carfilzomib (KYPROLIS®), cemiplimab-rwlc (LIBTAYO®), ceritinib (ZYKADIA®), cetuximab (ERBITUX®), ciltacabtagene autoleucel (CARVYKTI®), cobimetinib fumarate (COTELLIC®), copanlisib hydrochloride (ALIQUOPA®), crizotinib (XALKORI®), dabrafenib (TAFMLAR®), dabrafenib mesylate (TAFMLAR®), dacomitinib (VIZIMPRO®), daratumumab (DARZALEX®), daratumumab and hyaluronidase-fihj (DARZALEX FASPRO®), darolutamide (NUBEQA®), dasatinib (SPRYCEL®), denileukin diftitox (ONTAK®), denosumab (XGEVA®), dinutuximab (UNITUXIN®), dostarlimab-gxly (JEMPERLI®), durvalumab (IMFINZI®), duvelisib (COPIKTRA®), elacestrant dihydrochloride (ORSERDU®), elotuzumab (EMPLICITI®), enasidenib mesylate (IDHIFA®), encorafenib (BRAFTOVI®), enfortumab vedotin-ejfv (PADCEV®), entrectinib (ROZLYTREK®), enzalutamide (XTANDI®), erdafitinib (BAL VERSA®), erlotinib hydrochloride (TARCEVA®), everolimus (AFINITOR®), exemestane (AROMASIN®), famtrastuzumab deruxtecan-nxki (ENHERTU®), fedratinib hydrochloride (INREBIC®), fulvestrant (FASLODEX®), futibatinib (LYTGOBI®), gefitinib (IRESSA®), gemtuzumab ozogamicin (MYLOTARG®), gilteritinib fumarate (XOSPATA®), glasdegib maleate (DAURISMO®), ibritumomab tiuxetan (ZEVALIN®), ibrutinib (IMBRUVICA®), idecabtagene vicleucel (ABECMA®), idelalisib (ZYDELIG®), imatinib mesylate (GLEEVEC®), infigratinib phosphate (TRUSELTIQ®), inotuzumab ozogamicin (BESPONSA®), iobenguane 1 131 (AZEDRA®), ipilimumab (YERVOY®), isatuximab-irfc (SARCLISA®), ivosidenib (TIBSOVO®), ixazomib citrate (NINLARO®), lanreotide acetate (SOMATULINE DEPOT®), lapatinib ditosylate (TYK.ERB®), larotrectinib sulfate (VITRAKVI®), lenvatinib mesylate (LENVIMA®), letrozole (FEMARA®), lisocabtagene maraleucel (BREYANZI®), loncastuximab tesirine-lpyl (ZYNLONTA®), lorlatinib (LORBRENA®), lutetium Lu 177 vipivotide tetraxetan (PLUVICTO®), lutetium Lu 177-dotatate (LUTATHRA®), margetuximab- cmkb (MARGENZA®), midostaurin (R YD APT®), mirvetuximab soravtansine-gynx (ELAHERE®), mobocertinib succinate (EXKIVITY®), mogamulizumab-kpkc (POTELIGEO®), mosunetuzumab-axgb (LUNSUMIO®), moxetumomab pasudotox-tdfkAttorney Docket No.: GH0256WO(LUMOXITI®), naxitamab-gqgk (DANYELZA®), necitumumab (PORTRAZZA®), neratinib maleate (NERLYNX®), nilotinib (TASIGNA®), niraparib tosylate monohydrate (ZEJULA®), nivolumab (OPDIVO®), nivolumab and relatlimab-rmbw (OPDUALAG®), obinutuzumab (GAZYVA®), ofatumumab (ARZERRA®), olaparib (LYNPARZA®), olutasidenib (REZLHIDIA®), osimertinib mesylate (TAGRISSO®), pacritinib citrate (VONJO®), palbociclib (IBRANCE®), panitumumab (VECTIBIX®), pazopanib hydrochloride (VOTRIENT®), pembrolizumab (KEYTRUDA®), pemigatinib (PEMAZYRE®), pertuzumab (PERJETA®), pertuzumab, trastuzumab, and hyaluronidase-zzxf (PHESGO®), pexidartinib hydrochloride (TURALIO®), pirtobrutinib (JAYPIRCA®), polatuzumab vedotin-piiq (POLIVY®), ponatinib hydrochloride (ICLUSIG®), pralatrexate (FOLOTYN®), pralsetinib (GAVRETO®), radium 223 dichloride (XOFIGO®), ramucirumab (CYRAMZA®), regorafenib (STIVARGA®), retifanlimab-dlwr (ZYNYZ®), ribociclib (KISQALI®), ripretinib (QINLOCK®), rituximab (RITUXAN®), rituximab and hyaluronidase human (RITUXAN HYCELA®), romidepsin (ISTODAX®), rucaparib camsylate (RUBRACA®), ruxolitinib phosphate (JAKAFI®), sacituzumab govitecan-hziy (TRODELVY®), selinexor (XPOVIO®), selpercatinib (RETEVMO®), selumetinib sulfate (KOSELUGO®), siltuximab (SYLVANT®), sirolimus protein-bound particles (FYARRO®), sonidegib (ODOMZO®), sorafenib tosylate (NEXAVAR®), sotorasib (LUMAKRAS®), sunitinib malate (SUTENT®), tafasitamab-cxix (MONJUVI®), tagraxofusp-erzs (ELZONRIS®), talazoparib tosylate (TALZENNA®), tamoxifen citrate (SOLTAMOX®), tazemetostat hydrobromide (TAZVERIK®), tebentafusp- tebn (KIMMTRAK®), teclistamab-cqyv (TECVAYLI®), temsirolimus (TORISEL®), tepotinib hydrochloride (TEPMETKO®), tisagenlecleucel (KYMRIAH®), tisotumab vedotin-tftv (TIVDAK®), tivozanib hydrochloride (FOTIVDA®), toremifene (FARESTON®), trametinib (MEKINIST®), trametinib dimethyl sulfoxide (MEKINIST®), trastuzumab (HERCEPTIN®), tremelimumab-actl (IMJUDO®), tretinoin (VESANOID®), tucatinib (TUKYSA®), vandetanib (CAPRELSA®), vemurafenib (ZELBORAF®), venetoclax (VENCLEXTA®), vismodegib (ERIVEDGE®), vorinostat (ZOLINZA®), zanubrutinib (BRUKINSA®), and / or ziv-aflibercept (ZALTRAP®).

[0375] Table 1 provides an exemplary list of drugs used to treat cancers with mutations observed in target genes associated with certain cancer types. In certain embodiments, the subject has a cancer of a type listed in Table 1 including a mutation in one or more target genes listed in TableAttorney Docket No.: GH0256WO1 for that cancer type, and the therapy administered to the subject comprises the drug listed inTable 1 for that cancer type and mutation.Table 1. Exemplary drugsAttorney Docket No.: GH0256WOAttorney Docket No.: GH0256WOAttorney Docket No.: GH0256WOAttorney Docket No.: GH0256WOAttorney Docket No.: GH0256WOAttorney Docket No.: GH0256WOAttorney Docket No.: GH0256WOAttorney Docket No.: GH0256WO

[0376] In some embodiments, the methods described herein can be used to treat patients by (i) detecting one or more mutations in the one or more EV-associated target genes listed in Table 1; and (ii) administering the corresponding one or more drugs listed in Table 1. In some embodiments, these therapies may be used alone or in combination with other therapies to treat a disease.

[0377] These methods provided herein provide a deeper understanding of the changes in DNA and proteins that cause cancer, allowing the identification of biomarkers and design of treatments that target these proteins. In some embodiments, the biomarker may include an epigenetic signature, such as a methylation state, methylation score and / or DNA fragmentation pattem / score. In some embodiments, the epigenetic signature can be determined for one or more regions that include, but not limited to, transcription start sites, promoter regions, CTCF binding regions and regulatory protein binding regions. In some embodiments, the epigenetic signature is determined for one or more regions that include, but not limited to, transcription start sites, promoter regions, intergenic regions and / or intronic regions that are associated with at least one or more genes listed in Table 1. Such treatments may include small-molecule drugs or monoclonal antibodies. The methods may also improve biomarker testing in individuals suffering from disease and help determine if the individual is a candidate for a certain drug or combination of drugs based on the presence or absence of the biomarker. Additionally, the methods can improve identification of mutations that contribute to the development of resistance to targeted therapy. Consequently, the analysis techniques may reduce unnecessary or untimely therapeutic interventions, patient suffering, and patient mortality.

[0378] In certain embodiments, the immune checkpoint molecule is a co-stimulatory molecule that amplifies a signal involved in a T cell response to an antigen. For example, CD28 is a costimulatory receptor expressed on T cells. When a T cell binds to antigen through its T cell receptor, CD28 binds to CD80 (aka B7.1) or CD86 (aka B7.2) on antigen-presenting cells to amplify T cell receptor signaling and promote T cell activation. Because CD28 binds to the same ligands (CD80 and CD86) as CTLA4, CTLA4 is able to counteract or regulate the co-stimulatory signaling mediated by CD28. In certain embodiments, the immune checkpoint molecule is a co-stimulatory molecule selected from CD28, inducible T cell co-stimulator (ICOS), CD137, 0X40, or CD27. InAttorney Docket No.: GH0256WO other embodiments, the immune checkpoint molecule is a ligand of a co- stimulatory molecule, including, for example, CD80, CD86, B7RP1, B7-H3, B7-H4, CD137L, OX40L, or CD70.

[0379] Agonists that target these co-stimulatory checkpoint molecules can be used to enhance antigen-specific T cell responses against certain cancers. Accordingly, in certain embodiments, the immunotherapy or immunotherapeutic agent is an agonist of a co-stimulatory checkpoint molecule. In certain embodiments, the agonist of the co-stimulatory checkpoint molecule is an agonist antibody and preferably is a monoclonal antibody. In certain embodiments, the agonist antibody or monoclonal antibody is an anti-CD28 antibody. In other embodiments, the agonist antibody ormonoclonal antibody is an anti-ICOS, anti-CD137, anti-OX40, or anti-CD27 antibody. In other embodiments, the agonist antibody or monoclonal antibody is an anti-CD80, anti-CD86, anti-B7RPl, anti-B7-H3, anti-B7-H4, anti-CD137L, anti-OX40L, or anti-CD70 antibody.

[0380] In certain embodiments, the status of a nucleic acid variant from a sample from a subject as being of somatic or germline origin may be compared with a database of comparator results from a reference population to identify customized or targeted therapies for that subject. Typically, the reference population includes patients with the same cancer or disease type as the subject and / or patients who are receiving, or who have received, the same therapy as the subject. A customized or targeted therapy (or therapies) may be identified when the nucleic variant and the comparator results satisfy certain classification criteria (e.g., are a substantial or an approximate match).

[0381] In certain embodiments, the customized therapies described herein are typically administered parenterally (e.g., intravenously or subcutaneously). Pharmaceutical compositions containing an immunotherapeutic agent are typically administered intravenously. Certain therapeutic agents are administered orally. However, customized therapies (e.g., immunotherapeutic agents, etc.) may also be administered by any method known in the art, for example, buccal, sublingual, rectal, vaginal, intraurethral, topical, intraocular, intranasal, and / or intraauricular, which administration may include tablets, capsules, granules, aqueous suspensions, gels, sprays, suppositories, salves, ointments, or the like.

[0382] In some embodiments, therapy is customized based on the status of a nucleic acid variant as being of somatic or germline origin. In some embodiments, determination of the levels of particular cell types, e.g., immune cell types, including rare immune cell types, facilitates selection of appropriate treatment.10 / Attorney Docket No.: GH0256WO

[0383] The present methods can be used to diagnose the presence of a condition, e.g., cancer or precancer, in a subject, to characterize a condition (such as to determine a cancer stage or heterogeneity of a cancer), to monitor a subject’s response to receiving a treatment for a condition (such as a response to a chemotherapeutic or immunotherapeutic), assess prognosis of a subject (such as to predict a survival outcome in a subject having a cancer), to determine a subject’s risk of developing a condition, to predict a subsequent course of a condition in a subject, to determine metastasis or recurrence of a cancer in a subject (or a risk of cancer metastasis or recurrence), and / or to monitor a subject’s health as part of a preventative health monitoring program (such as to determine whether and / or when a subject is in need of further diagnostic screening). The methods according to the present disclosure can also be useful in predicting a subject’s response to a particular treatment option. Successful treatment options may increase the amount of EV-associated target molecules comprising proteins and / or nucleic acids exhibiting copy number variation, rare mutations, and / or cancer-related epigenetic signatures (such as hypermethylated regions or hypomethylated regions) detected in a subject's blood (such as in EVs isolated from a buffy coat sample or any other sample comprising cells, such as a blood sample (e.g., a whole blood sample, a buffy coat sample, a leukapheresis sample, or a PBMC sample) from the subject) if the treatment is successful as more cancer cells may die and shed or release EVs, and an unsuccessful treatment results in no change. In other examples, this may not occur. In another example, certain treatment options may be correlated with EV- associated transcriptomic or genetic profiles of cancers over time. This correlation may be useful in selecting a therapy for a subject. In some embodiments, determination of the metastasis site facilitates selection of appropriate treatment.

[0384] Thus, in some embodiments, quantities of each of one or more of a particular EV- associated proteomic, genetic and / or epigenetic signature (e.g., quantities of fusions, indels, SNPs, CNVs, and / or rare mutations, and / or cancer-related epigenetic signatures (such as specific (e.g., DMRs) or global hypermethylated or hypomethylated regions, and / or fragmentation variable regions)) in EV-associated DNA from a subject's blood (such as in EV-associated DNA from a blood sample (e.g., a whole blood sample) from the subject)) are determined based on sequencing and analysis. In some embodiments, quantities of each of a plurality of cell types, such as immune cell types, are determined based on sequencing and analysis of EV-associated target molecules isolated from at least one sample comprising cells (such as blood sample (e.g., aAttorney Docket No.: GH0256WO whole blood sample, a buffy coat sample, a leukapheresis sample, or a PBMC sample) from a subject. The plurality of immune cell types can include, but is not limited to, macrophages (including Ml macrophages and M2 macrophages), activated B cells (including regulatory B cells, memory B cells and plasma cells); T cell subsets, such as central memory T cells, naive- like T cells, and activated T cells (including cytotoxic T cells, regulatory T cells (Tregs), CD4 effector memory T cells, CD4 central memory T cells, CD8 effector memory T cells, and CD8 central memory T cells); immature myeloid cells (including myeloid-derived suppressor cells (MDSCs), low-density neutrophils, immature neutrophils, and immature granulocytes); and natural killer (NK) cells. As disclosed herein, differences in levels and / or presence of particular proteomic, genetic, and / or epigenetic signatures in EVs isolated from blood samples from a subject can be used to quantify cell types, such as immune cell types, within the sample. Thus, a comparison of one or more proteomic, genetic, and / or epigenetic signatures in EVs isolated from blood samples collected from a subject at two or more time points can be used to monitor changes in the one or more signatures and / or the one or more cell type quantities in the subject under different conditions (such as prior to and after a treatment), or over time (e.g., as part of a preventative health monitoring program).

[0385] In some embodiments, therapy is customized based on the status of a detected EV- associated nucleic acid variant as being of somatic or germline origin. In some embodiments, essentially any cancer therapy (e.g., surgical therapy, radiation therapy, chemotherapy, and / or the like) may be included as part of these methods. Typically, customized therapies include at least one immunotherapy (or an immunotherapeutic agent). Immunotherapy refers generally to methods of enhancing an immune response against a given cancer type. In certain embodiments, immunotherapy refers to methods of enhancing a T cell response against a tumor or cancer.

[0386] Therapies can function by helping the immune system destroy cancer cells. For example, certain targeted therapies may mark cancer cells for the immune system to destroy them. Other targeted therapies may support the immune system to work more effectively against cancer. Yet other therapies may stop cancer cells from growing, for example, by interfering with cancer cell surface markers preventing them from dividing. Additionally, therapies can inhibit signals that promote angiogenesis. Such angiogenesis inhibitors prevent blood supply into the tumor thereby, preventing tumor growth. Other targeted therapies can deliver toxic substances to the tumor.Attorney Docket No.: GH0256WOExamples include monoclonal antibodies combined with toxins, chemotherapy, or radiation. Some targeted therapies induce apoptosis or deplete cancer of hormones.

[0387] In certain embodiments, the status of an EV-associated nucleic acid variant from a sample from a subject as being of somatic or germline origin may be compared with a database of comparator results from a reference population to identify customized or targeted therapies for that subject. Typically, the reference population includes patients with the same cancer or disease type as the subject and / or patients who are receiving, or who have received, the same therapy as the subject. A customized or targeted therapy (or therapies) may be identified when the nucleic variant and the comparator results satisfy certain classification criteria (e.g., are a substantial or an approximate match).

[0388] The disclosed methods can include evaluating (such as quantifying) and / or interpreting a plurality of EV-associated target molecules released from a potential metastasis site (such as at least one cell material in a sample from a subject) and / or cell types that contribute to EVs, in one or more samples collected from a subject at one or more timepoints in comparison to a selected baseline value or reference standard (or a selected set of baseline values or reference standards). A baseline value or reference standard may be a presence or level of the plurality of EV- associated target molecules measured in one or more samples (such as an average quantity or range of quantities of cell types present in at least two samples) collected from the subject at one or more time points, such as prior to receiving a treatment, prior to diagnosis of a condition (such as a cancer), or as part of a preventative health monitoring program. A baseline value or reference standard may be a presence or level of the plurality of EV-associated target molecules measured with respect to one or more samples collected at one or more timepoints from one or more subjects that do not have the condition (such as a healthy subject that does not have a cancer), one or more subjects that responded favorably to the treatment, or one or more subjects that have not received the treatment.

[0389] The disclosed methods can include evaluating (such as quantifying) and / or interpreting one or more EV-associated target molecules present in one or more samples (e.g., in a blood sample (e.g., a whole blood sample, a buffy coat sample, a leukapheresis sample, or a PBMC sample)) collected from a subject at one or more timepoints in comparison to a selected baseline value or reference standard (or a selected set of baseline values or reference standards). A baseline value or reference standard may be a quantity of EV-associated target proteins, copyAttorney Docket No.: GH0256WO number variation, rare mutations, and / or cancer-related epigenetic signatures (such as hypermethylated regions or hypomethylated regions) measured in one or more samples (such as an average quantity or range of quantities of such signatures present in at least two samples) collected from the subject at one or more time points, such as prior to receiving a treatment, prior to diagnosis of a condition (such as a cancer), or as part of a preventative health monitoring program. A baseline value or reference standard may be a quantity of, e.g., EV-associated target proteins, copy number variation, rare mutations, and / or cancer-related epigenetic signatures (such as hypermethylated regions or hypomethylated regions) measured in one or more samples (such as an average quantity or range of quantities of such signatures and / or cell types present in at least two samples) collected at one or more timepoints from one or more subjects that do not have the condition (such as a healthy subject that does not have a cancer), one or more subjects that responded favorably to the treatment, or one or more subjects that have not received the treatment.

[0390] In certain embodiments, the baseline value or reference standard utilized is a standard or profile derived from a single reference subject. In other embodiments, the baseline value or reference standard utilized is a standard or profile derived from averaged data from multiple reference subjects. The reference standard, in various embodiments, can be a single value, a mean, an average, a numerical mean or range of numerical means, a numerical pattern, or a graphical pattern created from the genetic and / or epigenetic signature quantity data derived from a single reference subject or from multiple reference subjects. Selection of the particular baseline values or reference standards, or selection of the one or more reference subjects, depends upon the use to which the methods described herein are to be put by, for example, a research scientist or a clinician (such as a physician).

[0391] In some embodiments, one or more liquid biopsy samples (such as a buffy coat sample or any other sample comprising cells, such as a blood sample (e.g., a whole blood sample, a leukapheresis sample, or a PBMC sample) may be collected from a subject at two or more timepoints, to assess changes in EV-associated target molecules (such as changes in quantities of EV-associated target molecules) between the two timepoints. By monitoring EV-associated target molecules and identifying differences between samples collected from a subject at two or more timepoints, the present methods can be used, for example, to determine the presence or absence of a condition (such as a cancer), a response of the subject to a treatment, one or more1HAttorney Docket No.: GH0256WO characteristic of a condition (such as a cancer stage) in the subject, recurrence of a condition (such as a cancer), and / or a subject’s risk of developing a condition (such as a cancer). Thus, in some embodiments, methods are provided wherein quantities of EV-associated target molecules present in at least one sample (such as at least one whole blood sample, buffy coat sample, leukapheresis sample, or PBMC sample) collected from a subject at one or more timepoints (such as prior to receiving a treatment) are compared to quantities of EV-associated target molecules present in at least one sample collected from the subject at one or more different time points (such as after receiving the treatment). The disclosed methods can allow for patientspecific monitoring, such that, for example, differences in EV-associated target molecule quantities between samples collected from the subject at different timepoints may indicate changes (such as presence or absence of a condition, response to a treatment, a prognosis, or the like) that are significant with respect to the subject but may yet fall within a normal range of a general healthy population.

[0392] In some embodiments, methods are provided for monitoring a response (such as a change in disease state, such as a presence or absence of a metastasis in a subject, such as measured by assessing a presence or level of a plurality of EV-associated target molecules released from a potential metastasis site in a sample from the subject) of a subject to a treatment (such as a chemotherapy or an immunotherapy). In certain embodiments, one or more samples is collected from the subject at least 1-10, at least 1-5, at least 2-5, or at least 1, at least 2, least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 time points prior to the subject receiving the treatment. In certain embodiments, one or more samples is collected from the subject at least 1-10, at least 1-5, at least 2-5, or at least 1, at least 2, least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 time points after the subject has received the treatment. Sample collection from a subject can be ongoing during and / or after treatment to monitor the subject’s response to the treatment.

[0393] In some embodiments, samples are not collected from a subject prior to diagnosis of a condition (such as a cancer) or prior to receiving a treatment. In such embodiments, wherein the response of a subject to a treatment or the course or stage of a condition (such as a cancer) in the subject is being monitored over time, EV-associated target molecule signatures are compared between samples taken at least 2-10, at least 2-5, at least 3-6, or at least 2, such as at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20Attorney Docket No.: GH0256WO time points collected after the subject has been diagnosed and / or after the subject has received the treatment. Sample collection from a subject can be ongoing during and / or after treatment to monitor the subject’s response to the treatment.

[0394] In some embodiments of the disclosed methods, one or more samples is collected from a subject at least once per year, such as about 1-12 times or about 2-6 times, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times per year. In other embodiments, one or more samples is collected from the subject less than once per year, such as about once every 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months. In some embodiments, one or more samples is collected from the subject about once every 1-5 years or about once every 1-2 years, such as about every 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 years.

[0395] In other embodiments of the disclosed methods, one or more samples (such as one or more whole blood, buffy coat, leukapheresis, or PBMC samples) are collected from a subject at least once per week, such as on 1-4 days, 1-2 days, or on 1, 2, 3, 4, 5, 6, or 7 days per week. In certain embodiments, one or more samples are collected from the subject at least once per month, such as 1-15 times, 1-10 times, 2-5 times, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 times per month. In other embodiments, one or more samples is collected from the subject every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, or every 12 months. In some embodiments, one or more samples is collected from the subject at least once per day, such as 1, 2, 3, 4, 5, or 6 times per day. Selection of the one or more sample collection timepoints (e.g., the frequency of sample collection), or of the number of samples to be collected at each timepoint, depends upon the use to which the methods described herein are to be put by, for example, a research scientist or a clinician (such as a physician).IV. Kits

[0396] Also provided are kits comprising the compositions as described herein. The kits can be for use in performing the methods as described herein. In some embodiments, a kit comprises one or more target protein- and / or PTM-binding molecules. In some embodiments, the marker binding molecule comprises a label or capture moiety. In some embodiments, the kit comprises a solid support linked to a binding partner of the capture moiety. In some embodiments, the kit comprisesAttorney Docket No.: GH0256WO one or more target protein binding molecules. In some embodiments, the kit comprises reagents for detecting the presence or levels of target proteins.

[0397] In some embodiments, a kit further comprises an agent that recognizes methyl cytosine in DNA. In some such embodiments, the agent is an antibody or a methyl binding protein or methyl binding domain. In some embodiments, the kit comprises target-specific probes that specifically bind to epigenetic and / or sequence-variable target region sets. In some such embodiments, the target-specific probes comprise a capture moiety. In some embodiments, the kit comprises a solid support linked to a binding partner of the capture moiety. In some embodiments, the kit comprises adapters. In some embodiments, the kit comprises PCR primers, wherein the PCR primers anneal to a target region or to an adapter. In some embodiments, the kit comprises additional elements elsewhere herein. In some embodiments, the kit comprises instructions for performing a method described herein.

[0398] Kits may further comprise one or more immobilized EV marker binding molecules that are conjugated to a solid support. The immobilized EV marker binding molecules (such as immobilized antibodies) may hybridize to one or more or all of the EV-associated target molecules selected from CEA, Mucl, PD-L1, CD9, CD3, CD151, CD81, CD63, ALIX, CD40, Hsp60, TSG1, and phosphatidylserine.

[0399] Kits may further comprise a plurality of binding molecule-oligonucleotide conjugates (such as antibody-oligonucleotide conjugates) that selectively hybridize to one or more or all of the EV-associated target molecules selected from ALIX, CD40, TGS1, phosphatidyl serine, Mucl, CD3, CD 147, CD151, carcinoembryonic antigen (CEA), CD66a, CD326 (also known as epithelial cell adhesion molecule, EpCAM), CD49b, heat shock protein 60 (Hsp60), copine-3 (CPNE3), folate receptor alpha (FRa), CD24, vascular endothelial growth factor (VEGF), prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), epidermal growth factor receptor (EGFR), Glypican-3 (GPC-3), Glypican-1 (GPC-1), CD63, CD9, programmed death ligand 1 (PD- Ll), leucine-rich alpha-2 -glycoprotein 1 (LRG1), Epstein-Barr virus latent membrane protein 1 (LMP1), Epstein-Barr virus BamHI-A rightward frame 1 (BARF1), matrix metalloproteinase 9 (MMP9), ceruloplasmin (CP), Dickkopf-related protein 4 (DKK4), carbonic anhydrase 9 (CAIX), tumor-associated calcium signal transducer 2 (TACSTD2), CD36, CD44, Claudin 4, LI cell adhesion molecule (L1CAM), CD24, A disintegrin and metalloproteinase domain-containing protein 10 (ADAMI 0), extracellular matrix metalloproteinase inducer (EMMPRIN), transformingAttorney Docket No.: GH0256WO growth factor P-1 (TGFp-1), melanoma antigen 3 / 6 (MAGE-3 / 6), CD37, CD53, CD81 , CD47, CD26, CD196, CD274, CD133, and / or Ephrin A2. The kit can include a container that includes the plurality of binding molecule-oligonucleotide conjugates (such as antibody-oligonucleotide conjugates) and instructions for performing any of the methods described herein.

[0400] In some embodiments, each oligonucleotide conjugate comprises a first portion comprising a universal sequence region, a second portion comprising a unique sequence region that identifies an EV marker, and a third portion comprising a second universal sequence region. In some embodiments, the second universal sequence region comprises a double stranded portion and a single stranded portion.

[0401] Kits may further comprise a plurality of oligonucleotide probes that selectively hybridize to least 5, 6, 7, 8, 9, 10, 20, 30, 40 or all genes selected from the group consisting of ALK, APC, BRAF, CDKN2A, EGFR, ERBB2, FBXW7, KRAS, MYC, NOTCH1, NRAS, PIK3CA, PTEN, RBI, TP53, MET, AR, ABL1, AKT1, ATM, CDH1, CSFIR, CTNNB1, ERBB4, EZH2, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, MLH1, MPL, NPM1, PDGFRA, PROC, PTPN11, RET,SMAD4, SMARCB1, SMO, SRC, STK11, VHL, TERT, CCND1, CDK4, CDKN2B, RAFI, BRCA1, CCND2, CDK6, NF1, TP53, ARID 1 A, BRCA2, CCNE1, ESRI, RIT1, GATA3, MAP2K1, RHEB, ROS1, ARAF, MAP2K2, NFE2L2, RHOA, and NTRK1 . The number genes to which the oligonucleotide probes can selectively hybridize can vary. For example, the number of genes can comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54. The kit can include a container that includes the plurality of oligonucleotide probes and instructions for performing any of the methods described herein.

[0402] The kit can comprise at least 4, 5, 6, 7, or 8 different library adapters having distinct molecular barcodes and identical sample barcodes. The library adapters may not be sequencing adapters. For example, the library adapters do not include flow cell sequences or sequences that permit the formation of hairpin loops for sequencing. The different variations and combinations of molecular barcodes and sample barcodes are described throughout, and are applicable to the kit. Further, in some cases, the adapters are not sequencing adapters. Additionally, the adapters provided with the kit can also comprise sequencing adapters. A sequencing adapter can comprise a sequence hybridizing to one or more sequencing primers. A sequencing adapter can furtherAttorney Docket No.: GH0256WO comprise a sequence hybridizing to a solid support, e.g., a flow cell sequence. For example, a sequencing adapter can be a flow cell adapter. The sequencing adapters can be attached to one or both ends of a polynucleotide fragment. In some cases, the kit can comprise at least 8 different library adapters having distinct molecular barcodes and identical sample barcodes. The library adapters may not be sequencing adapters. The kit can further include a sequencing adapter having a first sequence that selectively hybridizes to the library adapters and a second sequence that selectively hybridizes to a flow cell sequence. In another example, a sequencing adapter can be hairpin shaped. For example, the hairpin shaped adapter can comprise a complementary double stranded portion and a loop portion, where the double stranded portion can be attached {e.g. , ligated) to a double-stranded polynucleotide. Hairpin shaped sequencing adapters can be attached to both ends of a polynucleotide fragment to generate a circular molecule, which can be sequenced multiple times. A sequencing adapter can comprise one or more barcodes. For example, a sequencing adapter can comprise a sample barcode. The sample barcode can comprise a predetermined sequence. The sample barcodes can be used to identify the source of the polynucleotides. The sample barcode can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more (or any length as described throughout) nucleic acid bases, e.g., at least 8 bases. The barcode can be contiguous or non-contiguous sequences, as described above.

[0403] The library adapters can be blunt ended and Y-shaped and can be less than or equal to 40 nucleic acid bases in length. Other variations of the can be found throughout and are applicable to the kit.V. Computer Systems

[0404] Methods of the present disclosure can be implemented using, or with the aid of, computer systems. FIG. 1 shows a computer system 101 that is programmed or otherwise configured to implement the methods of the present disclosure. The computer system 101 can regulate various aspects sample preparation, sequencing, and / or analysis. In some examples, the computer system 101 is configured to perform sample preparation and sample analysis, including (where applicable) nucleic acid sequencing, e.g., according to any of the methods disclosed herein.

[0405] The computer system 101 includes a central processing unit (CPU, also "processor" and "computer processor" herein) 105, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 101 also includes memoryAttorney Docket No.: GH0256WO or memory location 110 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 115 (e.g., hard disk), communication interface 120 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 125, such as cache, other memory, data storage, and / or electronic display adapters. The memory 110, storage unit 115, interface 120, and peripheral devices 125 are in communication with the CPU 105 through a communication network or bus (solid lines), such as a motherboard. The storage unit 115 can be a data storage unit (or data repository) for storing data. The computer system 101 can be operatively coupled to a computer network 130 with the aid of the communication interface 120. The computer network 130 can be the Internet, an internet and / or extranet, or an intranet and / or extranet that is in communication with the Internet. The computer network 130 in some cases is a telecommunication and / or data network. The computer network 130 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The computer network 130, in some cases with the aid of the computer system 101, can implement a peer-to-peer network, which may enable devices coupled to the computer system 101 to behave as a client or a server.

[0406] The CPU 105 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 110. Examples of operations performed by the CPU 105 can include fetch, decode, execute, and writeback.

[0407] The storage unit 115 can store files, such as drivers, libraries, and saved programs. The storage unit 115 can store programs generated by users and recorded sessions, as well as output(s) associated with the programs. The storage unit 115 can store user data, e.g., user preferences and user programs. The computer system 101 in some cases can include one or more additional data storage units that are external to the computer system 101, such as located on a remote server that is in communication with the computer system 101 through an intranet or the Internet. Data may be transferred from one location to another using, for example, a communication network or physical data transfer (e.g., using a hard drive, thumb drive, or other data storage mechanism).

[0408] The computer system 101 can communicate with one or more remote computer systems through the network 130. For embodiment, the computer system 101 can communicate with a remote computer system of a user (e.g., operator). Examples of remote computer systems include inAttorney Docket No.: GH0256WO personal computers (e.g., portable PC), slate or tablet PC's (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 101 via the network 130.

[0409] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 101, such as, for example, on the memory 110 or electronic storage unit 115. The machine executable or machine-readable code can be provided in the form of software. During use, the code can be executed by the processor 105. In some cases, the code can be retrieved from the storage unit 115 and stored on the memory 110 for ready access by the processor 105. In some situations, the electronic storage unit 115 can be precluded, and machine-executable instructions are stored on memory 110.

[0410] In an aspect, the present disclosure provides a non-transitory computer-readable medium comprising computer-executable instructions which, when executed by at least one electronic processor, perform at least a portion of a method described herein. For example, the method may comprise:

[0411] The code can be pre-compiled and configured for use with a machine have a processer adapted to execute the code or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as- compiled fashion.

[0412] Aspects of the systems and methods provided herein, such as the computer system 101, can be embodied in programming. Various aspects of the technology may be thought of as "products" or "articles of manufacture" typically in the form of machine (or processor) executable code and / or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. "Storage" type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time...

Claims

Attorney Docket No.: GH0256WOWhat is claimed is:

1. A method of analyzing extracellular vesicle (EV) markers in a sample, the method comprising: a) obtaining an EV-containing sample; b) enriching for EVs comprising a first EV marker using an immobilized first EV marker binding molecule that binds to the first EV marker, thereby obtaining immobilized EVs; c) contacting the immobilized EVs with a set of second EV marker binding molecule- oligonucleotide conjugates to generate complexes of second EV marker binding molecules bound to the immobilized EVs; d) removing unbound second EV marker binding molecule-oligonucleotide conjugates from the complexes of second EV marker binding molecules bound to the immobilized EVs; and e) detecting the second EV marker binding molecules bound to the immobilized EVs.

2. A method of analyzing EV markers in a sample, the method comprising: a) obtaining an EV-containing sample; b) enriching for EVs comprising a first EV marker using an immobilized first EV marker binding molecule that binds to the first EV marker, thereby obtaining immobilized EVs; c) contacting the immobilized EVs with a set of second EV marker binding molecule- oligonucleotide conjugates to generate complexes of second EV marker binding molecules bound to the immobilized EVs; d) releasing the immobilized EVs, thereby providing released EVs; e) removing unbound second EV marker binding molecule-oligonucleotide conjugates from the complexes of second EV marker binding molecules bound to the released EVs using chromatographic purification; and f) detecting the second EV marker binding molecules bound to the released EVs.

3. The method of any one of the preceding claims, wherein the EV markers are surface protein markers.

4. The method of any one of the preceding claims, wherein the set of second EV marker binding molecule-oligonucleotide conjugates comprises 2-100 EV marker bindingAttorney Docket No.: GH0256WO molecule-oligonucleotide conjugates, optionally wherein the set of second EV marker binding molecule-oligonucleotide conjugates comprises 2-100, 10-50, 20-40, 25-35, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more than 60 EV marker binding molecule- oligonucleotide conjugates.

5. The method of any one of the preceding claims, wherein one or more of the EV marker binding molecules comprise an antibody, a knottin, an aptamer, an affimer, an avimer, a nanobody, a DARPin, a monobody, or an affinity clamp.

6. The method of any one of the preceding claims, wherein one or more of the EV marker binding molecules comprise an antibody.

7. The method of any one of the preceding claims, wherein the EV marker binding molecule- oligonucleotide conjugates independently comprise an oligonucleotide comprising a first portion comprising a universal sequence region, a second portion comprising a unique sequence region that identifies an EV marker, and a third portion comprising a second universal sequence region.

8. The method of the immediately preceding claim, wherein the second universal sequence region comprises a double stranded portion and a single stranded portion.

9. The method of any one of claims 7-8, wherein the second universal sequence region comprises the 3’ end of the first oligonucleotide.

10. The method of any one of claims 8-9, wherein the single stranded portion of the second universal sequence region is located 3’ of the double stranded portion of the second universal sequence region.

11. The method of any one of claims 8-10, wherein the unique sequence region is distal to the single stranded portion of the second universal sequence region relative to the double stranded portion of the second universal sequence region.

12. The method of any one of claims 7-11, wherein the unique sequence region comprises a molecular tag.

13. The method of the immediately preceding claim, wherein the molecular tag is a molecular barcode.

14. The method of any one of the preceding claims, wherein the immobilized first EV marker binding molecule is bound to a solid support.Attorney Docket No.: GH0256WO15. The method of the immediately preceding claim, wherein a capture nucleotide sequence is linked to the solid support.

16. The method of the immediately preceding claim, wherein the capture nucleotide sequence can hybridize with a universal region of an EV marker binding molecule-oligonucleotide conjugate and thereby participate in proximity extension reactions.

17. The method of the immediately preceding claim, wherein the proximity extension reaction yields an extension product, further wherein the extension product is amplified by PCR.

18. The method any one of the preceding claims, wherein the detecting comprises high multiplex PCR, digital PCR, quantitative PCR (qPCR), reverse transcription PCR (RT- PCR), or sequencing.

19. The method of any one of the preceding claims, further comprising amplifying at least a portion of the oligonucleotides in the bound second EV marker binding molecule- oligonucleotide conjugates.

20. The method of the immediately preceding claim, wherein the detecting comprises sequencing at least a portion of the amplified oligonucleotides.

21. The method of the immediately preceding claim, wherein the sequencing comprises next generation sequencing (NGS).

22. The method of any one of the preceding claims, wherein the sample is partitioned into a plurality of subsamples prior to step (b).

23. The method of the immediately preceding claim, wherein steps (b)-(d) are performed in parallel for each subsample of the plurality of subsamples.

24. The method of any one of the preceding claims, wherein (a) obtaining an EV-containing sample comprises obtaining a plurality of EV-containing samples, and wherein the method further comprises performing steps (b)-(d) in parallel for each sample of the plurality of samples.

25. The method of the immediately preceding claim, wherein each EV-containing sample is obtained from a different tissue.

26. The method of any one of claims 24-25, wherein the first EV marker is a tissue-specific marker and / or a cell type marker.

27. The method of any one of claims 22-25, wherein a multiplexed sequencing is performed.Attorney Docket No.: GH0256WO28. The method of any one of claims 19-27, wherein a sample index is added in the amplification step.

29. The method of any one of claims 19-28, wherein a sequencing adapter is added in the amplification step.

30. The method of any one of claims 19-29, wherein a molecular tag specific to each EV marker binding molecule is added in the amplification step.

31. The method of any one of the preceding claims, wherein the first EV marker is selected from CEA, Mucl, PD-L1, CD9, CD3, CD151, CD81, CD63, ALIX, CD40, Hsp60, TSG1, and phosphatidylserine.

32. The method of any one of the preceding claims, wherein the second EV marker binding molecules bind to one or more of ALIX, CD40, TGS1, phosphatidylserine, Mucl, CD3, CD147, CD151, carcinoembryonic antigen (CEA), CD66a, CD326 (also known as epithelial cell adhesion molecule, EpCAM), CD49b, heat shock protein 60 (Hsp60), copine-3 (CPNE3), folate receptor alpha (FRa), CD24, vascular endothelial growth factor (VEGF), prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), epidermal growth factor receptor (EGFR), Glypican-3 (GPC-3), Glypican-1 (GPC-1), CD63, CD9, programmed death ligand 1 (PD-L1), leucine-rich alpha-2-glycoprotein 1 (LRG1), Epstein-Barr virus latent membrane protein 1 (LMP1), Epstein-Barr virus BamHI-A rightward frame 1 (BARF1), matrix metalloproteinase 9 (MMP9), ceruloplasmin (CP), Dickkopf-related protein 4 (DKK4), carbonic anhydrase 9 (CAIX), tumor-associated calcium signal transducer 2 (TACSTD2), CD36, CD44, Claudin 4, LI cell adhesion molecule (L1CAM), CD24, A disintegrin and metalloproteinase domaincontaining protein 10 (ADAMI 0), extracellular matrix metalloproteinase inducer (EMMPRIN), transforming growth factor P-1 (TGF -1), melanoma antigen 3 / 6 (MAGE- 3 / 6), CD37, CD53, CD81, CD47, CD26, CD196, CD274, CD133 , and / or Ephrin A2.

33. The method of any one of the preceding claims, wherein the second EV marker binding molecules bind to 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, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more,Attorney Docket No.: GH0256WO36 or more, 37 or more, 38 or more, 39 or more, 40 or more, 41 or more, 42 or more, 43 or more, 44 or more, 45 or more, 46 or more, 47 or more, 48 or more, 49 or more, 50 or more, or each of ALIX, CD40, TGS1, phosphatidylserine, Mucl, CD3, CD147, CD151, carcinoembryonic antigen (CEA), CD66a, CD326 (also known as epithelial cell adhesion molecule, EpCAM), CD49b, heat shock protein 60 (Hsp60), copine-3 (CPNE3), folate receptor alpha (FRa), CD24, vascular endothelial growth factor (VEGF), prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), epidermal growth factor receptor (EGFR), Glypican-3 (GPC-3), Glypican-1 (GPC-1), CD63, CD9, programmed death ligand 1 (PD-L1), leucine-rich alpha-2-glycoprotein 1 (LRG1), Epstein-Barr virus latent membrane protein 1 (LMP1), Epstein-Barr virus BamHI-A rightward frame 1 (BARF1), matrix metalloproteinase 9 (MMP9), ceruloplasmin (CP), Dickkopf-related protein 4 (DKK4), carbonic anhydrase 9 (CAIX), tumor-associated calcium signal transducer 2 (TACSTD2), CD36, CD44, Claudin 4, LI cell adhesion molecule (L1CAM), CD24, A disintegrin and metalloproteinase domain-containing protein 10 (ADAM 10), extracellular matrix metalloproteinase inducer (EMMPRIN), transforming growth factor P-1 (TGF -1), melanoma antigen 3 / 6 (MAGE-3 / 6), CD37, CD53, CD81, CD47, CD26, CD196, CD274, CD133, and / or Ephrin A2.

34. The method of any one of the preceding claims, wherein the sample is a urine sample, an ascites sample, or a saliva sample.

35. The method of any one of the preceding claims, wherein the sample is a blood sample.

36. The method of the immediately preceding claim, wherein the blood sample is a whole blood sample, a plasma sample, a buffy coat sample, a leukapheresis sample, or a PBMC sample.

37. The method of any one of the preceding claims, wherein the sample comprises plasma obtained from a blood sample.

38. The method of any one of the preceding claims, wherein the sample comprises serum.

39. The method of any one of the preceding claims, wherein the sample is a tissue sample.

40. The method of the immediately preceding claim, wherein the tissue sample is a biopsy, a fine needle aspirate, or a formalin-fixed paraffin-embedded tissue sample.

41. The method of any one of the preceding claims, wherein at least one of the EV markers is a cell type marker.Attorney Docket No.: GH0256WO42. The method of any one of the preceding claims, wherein the detecting step further comprises quantifying the level of one or more of the EV markers present in the sample.

43. The method of any one of the preceding claims, wherein the sample is obtained from a subject.

44. The method of the immediately preceding claim, wherein the subject is an animal.

45. The method of any one of claims 43-44, wherein the subject is a human.

46. The method of any one of the preceding claims, wherein at least one of the EV markers is associated with a disease or condition.

47. The method of the immediately preceding claim, wherein the disease or condition is a cancer.

48. The method of any one of claims 46-47, wherein the subject has or is at risk of having the disease or condition.

49. The method of any one of claims 43-48, wherein the method comprises analyzing EV markers in a subsample of the sample or in a second sample obtained from the same subject from which the first sample is obtained.

50. The method of any one of claims 43-49, comprising determining a likelihood that the subject has precancer.

51. The method of any one of claims 43-50, comprising determining a likelihood that the subject has cancer.

52. The method of any one of the preceding claims, wherein the sample is obtained from a subject who was previously diagnosed with a cancer and received one or more previous cancer treatments, optionally wherein the sample is obtained at one or more preselected time points following the one or more previous cancer treatments.

53. The method of the immediately preceding claim, further comprising determining a cancer recurrence score, optionally wherein the cancer recurrence status of the subject is determined to be at risk for cancer recurrence when a cancer recurrence score is determined to be at or above a predetermined threshold or the cancer recurrence status of the subject is determined to be at lower risk for cancer recurrence when the cancer recurrence score is below the predetermined threshold.

54. The method of the immediately preceding claim, further comprising comparing the cancer recurrence score of the subject with a predetermined cancer recurrence threshold, whereinAttorney Docket No.: GH0256WO the subject is classified as a candidate for a subsequent cancer treatment when the cancer recurrence score is above the cancer recurrence threshold or not a candidate for a subsequent cancer treatment when the cancer recurrence score is below the cancer recurrence threshold.

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