Methods for detecting extracellular vesicle-associated molecules

Abstract

Provided herein are methods of analyzing a plurality of extracellular vesicle (EV)-associated target molecules in a sample. An exemplary method comprises (a) partitioning extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles; and (b) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules using nucleic acid sequencing.

Description

Atty. Docket No. GH0250WO METHODS FOR DETECTING EXTRACELLULAR VESICLE-ASSOCIATED MOLECULES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of US Provisional Patent Application No.63 / 721,181, filed November 15, 2024, which is incorporated by reference herein in its entirety for all purposes.FIELD OF THE INVENTION

[0002] The present disclosure provides methods related to analyzing a plurality of extracellular vesicle (EV)-associated target molecules in a sample, such as a liquid biopsy sample, such as a blood sample. In some embodiments, extracellular vesicles in the sample are partitioned into a plurality of compartments such that only a single extracellular vesicle is present in at least a portion of the plurality of compartments. In some embodiments, for at least a portion of the partitioned extracellular vesicles, a presence, absence, or level of each of the plurality of EV-associated target molecules are detected. In some embodiments, optionally prior to the partitioning, the sample is contacted with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule. In some embodiments, the sample is from a subject having or suspected of having cancer.INTRODUCTION AND SUMMARY

[0003] Cancer is responsible for millions of deaths per year worldwide. Early cancer detection may result in improved outcomes because early-stage cancer tends to be more susceptible to treatment.

[0004] Improperly controlled cell growth is a hallmark of cancer. Cancer is usually caused by the accumulation of mutations within an individual's normal cells, at least some resulting in improperly regulated cell division. Such mutations commonly include single nucleotide variations (SNVs), gene fusions, insertions and deletions (indels), transversions, translocations, and inversions. Cancers may also exhibit an accumulation of epigenetic changes, including modification of cytosine (e.g., 5-methylcytosine, 5-hydroxymethylcytosine, and other more oxidized forms) and association of DNA with chromatin proteins and transcription factors.Atty. Docket No. GH0250WO

[0005] Biopsies represent a traditional approach for detecting or diagnosing cancer in which cells or tissue are extracted from a possible cancer site and analyzed for relevant phenotypic and / or genotypic features. Biopsies have the drawback of being invasive.

[0006] Cancer detection based on analysis of body fluids (“liquid biopsies”), such as blood, urine, ascites, or saliva, is an intriguing alternative based on the observation that cancer-associated molecules and cellular components, such as cancer-associated extracellular vesicles, may be present in body fluids. A liquid biopsy is noninvasive (sometimes requiring only a blood draw). However, it has been challenging to develop accurate and sensitive methods for analyzing liquid biopsy material because the amount of cancer-associated molecules and cellular components released into body fluids is low and variable, as is recovery of, e.g., nucleic acids or proteins from such fluids in analyzable form. These sources of variation can obscure the predictive value of mutations (e.g., rearrangements, such as translocations and indels) among samples. Such mutations may include biomarkers that can be used to evaluate whether a subject diagnosed with, or suspected of having signs of, a cancer will benefit from a specific type of cancer therapy, such as Immuno-Oncology (I-O) therapy. Isolating and processing extracellular vesicle (EV)-associated molecules useful for further analysis in liquid biopsy procedures can be a useful part of these methods. EVs and their associated molecules mauy be useful as biomarkers of disease. Accordingly, there is a need for improved methods and compositions for analyzing EV-associated molecules, e.g., in liquid biopsies, such as at the single-EV level. Such methods are needed to improve the general understanding of heterogenous EV populations, to permit identification of unique subpopulations, and to enable earlier and more sensitive disease detection.

[0007] The methods herein can provide information about EV-associated molecules, including but not limited to EV-associated proteins and EV-associated nucleic acid sequences. Existing methods may not provide capture and multiplex analysis of proteins (such as cancer markers) and / or nucleic acid sequences (such as cancer-related sequences) associated with individual EVs in a sample.

[0008] The present disclosure aims to meet the need for improved analysis of EV-associated molecules (such as cancer-related molecules), such as using amplification and / or sequencing techniques, provide other benefits, or at least provide the public with a useful choice.Accordingly, the following exemplary embodiments are provided.Atty. Docket No. GH0250WO

[0009] Embodiment l is a method of analyzing a plurality of extracellular vesicle (EV)-associated target molecules in a sample, the method comprising:a) partitioning extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles; andb) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules using nucleic acid sequencing;optionally wherein the sample is contacted with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agentspecific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule, further optionally wherein the contacting occurs prior to step (a).

[0010] Embodiment 1.1 is a method of analyzing a plurality of extracellular vesicle (EV)-associated target molecules in a sample, the method comprising:a) partitioning extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles; andb) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules using nucleic acid sequencing;optionally wherein the sample is contacted with a plurality of antibody-oligonucleotide conjugates comprising at least (i) a first antibody-oligonucleotide conjugate comprising a first oligonucleotide and a first antibody specific for a first EV-associated target molecule, and (ii) a second antibody-oligonucleotide conjugate comprising a second oligonucleotide and a second antibody specific for a second EV-associated target molecule, further optionally wherein the contacting occurs prior to step (a).

[0011] Embodiment 2 is a method of analyzing a plurality of extracellular vesicle (EV)-associated target molecules in a sample, the method comprising:a) contacting the sample with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a firstAtty. Docket No. GH0250WOoligonucleotide and a first binding agent specific for a first EV-associated target molecule, (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule, (iii) a third binding agent-oligonucleotide conjugate comprising a third oligonucleotide and a third binding agentspecific for a third EV-associated target molecule, and (iv) a fourth binding agent-oligonucleotide conjugate comprising a fourth oligonucleotide and a fourth binding agent specific for a fourth EV-associated target molecule;b) partitioning the extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles; and c) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules by amplifying and / or sequencing oligonucleotides of the plurality of binding agent-oligonucleotide conjugates.

[0012] Embodiment 2.1 is a method of analyzing a plurality of extracellular vesicle (EV)-associated target molecules in a sample, the method comprising:a) contacting the sample with a plurality of antibody-oligonucleotide conjugates comprising at least (i) a first antibody-oligonucleotide conjugate comprising a first oligonucleotide and a first antibody specific for a first EV-associated target molecule, (ii) a second antibody-oligonucleotide conjugate comprising a second oligonucleotide and a second antibody specific for a second EV-associated target molecule, (iii) a third antibody-oligonucleotide conjugate comprising a third oligonucleotide and a third antibody specific for a third EV-associated target molecule, and (iv) a fourth antibody-oligonucleotide conjugate comprising a fourth oligonucleotide and a fourth antibody specific for a fourth EV-associated target molecule; b) partitioning the extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles; and c) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules by amplifying and / or sequencing oligonucleotides of the plurality of antibody-oligonucleotide conjugates.

[0013] Embodiment 3 is a method of analyzing a plurality of extracellular vesicle (EV)-associated target molecules in a sample, the method comprising:Atty. Docket No. GH0250WOa) partitioning the extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles; and b) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules, wherein the plurality of EV-associated target molecules comprise a plurality of EV-associated nucleic acids, wherein the detecting comprises nucleic acid amplification and / or sequencing;optionally wherein the sample is contacted with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule, further optionally wherein the contacting occurs prior to step (a).

[0014] Embodiment 3.1 is a method of analyzing a plurality of extracellular vesicle (EV)-associated target molecules in a sample, the method comprising:a) partitioning the extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles; and b) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules, wherein the plurality of EV-associated target molecules comprise a plurality of EV-associated nucleic acids, wherein the detecting comprises nucleic acid amplification and / or sequencing;optionally wherein the sample is contacted with a plurality of antibody-oligonucleotide conjugates comprising at least (i) a first antibody-oligonucleotide conjugate comprising a first oligonucleotide and a first antibody specific for a first EV-associated target molecule, and (ii) a second antibody-oligonucleotide conjugate comprising a second oligonucleotide and a second antibody specific for a second EV-associated target molecule, further optionally wherein the contacting occurs prior to step (a).

[0015] Embodiment 4 is a method of analyzing a plurality of extracellular vesicle (EV)-associated target molecules in a sample, the method comprising:a) contacting the sample with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a firstAtty. Docket No. GH0250WO oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule; wherein the first oligonucleotide comprises a subsequence complementary to a first portion of the second oligonucleotide;b) partitioning extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles;c) before or after the partitioning, contacting the first and second oligonucleotides with a polymerase, wherein first and second oligonucleotides of binding agent-oligonucleotide conjugates that are bound to EV-associated target molecules of the same extracellular vesicle undergo extension to form extended oligonucleotides; andd) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules using amplification of the extended oligonucleotides.

[0016] Embodiment 4.1 is a method of analyzing a plurality of extracellular vesicle (EV)-associated target molecules in a sample, the method comprising:a) contacting the sample with a plurality of antibody-oligonucleotide conjugates comprising at least (i) a first antibody-oligonucleotide conjugate comprising a first oligonucleotide and a first antibody specific for a first EV-associated target molecule, and (ii) a second antibody-oligonucleotide conjugate comprising a second oligonucleotide and a second antibody specific for a second EV-associated target molecule; wherein the first oligonucleotide comprises a subsequence complementary to a first portion of the second oligonucleotide;b) partitioning extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles;c) before or after the partitioning, contacting the first and second oligonucleotides with a polymerase, wherein first and second oligonucleotides of antibody-oligonucleotide conjugates that are bound to EV-associated target molecules of the same extracellular vesicle undergo extension to form extended oligonucleotides; andd) for at least a portion of the partitioned extracellular vesicles, detecting a presence,Atty. Docket No. GH0250WO absence, or level of each of the plurality of EV-associated target molecules using amplification of the extended oligonucleotides.

[0017] Embodiment 5 is a method of analyzing a plurality of extracellular vesicle (EV)-associated target molecules in a sample, the method comprising:a) contacting the sample with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule; wherein the first oligonucleotide comprises a first subsequence complementary to a first portion of a connector oligonucleotide and the second oligonucleotide comprises a second subsequence complementary to a second portion of the connector oligonucleotide;b) partitioning extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles;c) before or after the partitioning, contacting the first and second oligonucleotides with a ligase in the presence of the connector oligonucleotides, wherein first and second oligonucleotides of binding agent-oligonucleotide conjugates that are bound to EV-associated target molecules of the same extracellular vesicle undergo ligation to form ligated oligonucleotides; andd) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules using amplification of the ligated oligonucleotides.

[0018] Embodiment 5.1 is a method of analyzing a plurality of extracellular vesicle (EV)-associated target molecules in a sample, the method comprising:a) contacting the sample with a plurality of antibody-oligonucleotide conjugates comprising at least (i) a first antibody-oligonucleotide conjugate comprising a first oligonucleotide and a first antibody specific for a first EV-associated target molecule, and (ii) a second antibody-oligonucleotide conjugate comprising a second oligonucleotide and a second antibody specific for a second EV-associated target molecule; wherein the first oligonucleotide comprises a first subsequence complementary to a first portion of a connector oligonucleotide and the second oligonucleotide comprises a second subsequence complementary to a second portion of theAtty. Docket No. GH0250WO connector oligonucleotide;b) partitioning extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles;c) before or after the partitioning, contacting the first and second oligonucleotides with a ligase in the presence of the connector oligonucleotides, wherein first and second oligonucleotides of antibody-oligonucleotide conjugates that are bound to EV-associated target molecules of the same extracellular vesicle undergo ligation to form ligated oligonucleotides; andd) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules using amplification of the ligated oligonucleotides.

[0019] Embodiment 6 is the method of any one of the preceding embodiments, wherein the plurality of binding agent-oligonucleotide conjugates further comprises at least a third binding agent-oligonucleotide conjugate comprising a third binding agent specific for a third EV-associated target molecule.

[0020] Embodiment 7 is the method of the immediately preceding embodiment, wherein the plurality of binding agent-oligonucleotide conjugates further comprises at least a fourth binding agent-oligonucleotide conjugate comprising a fourth binding agent specific for a fourth EV-associated target molecule.

[0021] Embodiment 8 is the method of the immediately preceding embodiment, wherein the plurality of binding agent-oligonucleotide conjugates further comprises at least a fifth binding agent-oligonucleotide conjugate comprising a fifth binding agent specific for a fifth EV-associated target molecule.

[0022] Embodiment 9 is the method of any one of the preceding embodiments, wherein the plurality of binding agent-oligonucleotide conjugates comprises 2-30, such as 2-25, 2-20, 2-15, 2-10, 2-5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more than 30 binding agent-oligonucleotide conjugates.

[0023] Embodiment 10 is the method of any one of the preceding embodiments, wherein the binding agent comprises an antibody, a knottin, an aptamer, an affimer, an avimer, a nanobody, a DARPin, a monobody, or an affinity clamp.Atty. Docket No. GH0250WO

[0024] Embodiment 11 is the method of any one of the preceding embodiments, the binding agent comprises an antibody.

[0025] Embodiment 12 is the method of any one of the preceding embodiments, wherein the plurality of EV-associated target molecules comprises 2-30, such as 2-25, 2-20, 2-15, 2-10, 2-5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more than 30 EV-associated target molecules.

[0026] Embodiment 13 is the method of any one of the preceding embodiments, wherein the plurality of EV-associated target molecules comprises one or more proteins.

[0027] Embodiment 14 is the method of the immediately preceding embodiment, wherein the one or more proteins comprise one or more extracellular vesicle surface proteins.

[0028] Embodiment 15 is the method of any one of the preceding embodiments, wherein the plurality of EV-associated target molecules comprises one or more EV-associated target nucleic acids.

[0029] Embodiment 16 is the method of the immediately preceding embodiment, wherein the plurality of EV-associated target nucleic acids comprises one or more DNAs.

[0030] Embodiment 17 is the method of embodiment 15 or 16, wherein the plurality of EV-associated target nucleic acids comprises one or more RNAs.

[0031] Embodiment 18 is the method of the immediately preceding embodiment, wherein the one or more RNAs comprise one or more messenger RNAs (mRNA).

[0032] Embodiment 19 is the method of any one of embodiments 17-18, wherein the one or more RNAs comprise one or more ribosomal RNAs (rRNAs).

[0033] Embodiment 20 is the method of any one of embodiments 17-19, wherein the one or more RNAs comprise one or more transfer RNAs (tRNAs).

[0034] Embodiment 21 is the method of any one of embodiments 17-20, wherein the one or more RNAs comprise one or more microRNAs (miRNAs).

[0035] Embodiment 22 is the method of any one of embodiments 17-21, wherein the one or more RNAs comprise one or more small nuclear RNAs (snRNAs).

[0036] Embodiment 23 is the method of any one of embodiments 17-22, wherein the one or more RNAs comprise one or more small interfering RNAs (siRNAs).

[0037] Embodiment 24 is the method of any one of embodiments 17-23, wherein the one or more RNAs comprise one or more long non-coding RNAs (IncRNAs).

[0038] Embodiment 25 is the method of any one of embodiments 17-24, wherein the method comprises amplifying the plurality of EV-associated target nucleic acids.Atty. Docket No. GH0250WO

[0039] Embodiment 26 is the method of any one of embodiments 17-25, wherein one or more barcodes are added to the plurality of EV-associated target nucleic acids or amplification products thereof.

[0040] Embodiment 27 is the method of any one of the preceding embodiments wherein the one or more oligonucleotides comprises at least one tag.

[0041] Embodiment 28 is the method of the immediately preceding embodiment, wherein the at least one tag comprises a molecular barcode.

[0042] Embodiment 29 is the method of any one of the preceding embodiments, wherein the method comprises amplifying the one or more oligonucleotides.

[0043] Embodiment 30 is the method of embodiments 25-29, wherein the amplifying comprises PCR, reverse transcription PCR (RT-PCR), rolling circle amplification, or linear amplification.

[0044] Embodiment 31 is the method of any one of embodiments 25-30, wherein the amplifying occurs after the partitioning and prior to the detecting.

[0045] Embodiment 32 is the method of any one of the preceding embodiments, wherein the plurality of compartments is a plurality of droplets.

[0046] Embodiment 33 is the method of any one of the preceding embodiments, wherein the detecting comprises digital PCR, quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), sequencing, proximity ligation assay, and / or proximity extension assay.

[0047] Embodiment 34 is the method of any one of the preceding embodiments, wherein the detecting comprises proximity ligation assay or proximity extension assay, and wherein at least one of the plurality of binding agent-oligonucleotide conjugates comprises an binding agent specific for at least one extracellular vesicle-specific marker.

[0048] Embodiment 35 is the method of the immediately preceding embodiment, wherein the at least one extracellular vesicle-specific marker comprises one or more of CD81, CD63, CD9, ALIX, CD40, Hsp60, TSG1, or phosphatidyl serine.

[0049] Embodiment 36 is the method of the immediately preceding embodiment, wherein the digital PCR is droplet digital PCR.

[0050] Embodiment 37 is the method of any one of the preceding embodiments, wherein the sequencing comprises next generation sequencing.

[0051] Embodiment 38 is the method of any one of embodiments the preceding embodiments, wherein the sequencing comprises single-cell sequencing.Atty. Docket No. GH0250WO

[0052] Embodiment 39 is the method of any one of the preceding samples, comprising isolating at least a portion of the extracellular vesicles in the sample from other components of the sample, thereby providing isolated extracellular vesicles.

[0053] Embodiment 40 is the method of the immediately preceding embodiment, wherein the isolating comprises affinity purification, immunoprecipitation, size-exclusion chromatography, or centrifugation.

[0054] Embodiment 41 is the method of any one of embodiments 39-40, wherein the isolating comprises capturing extracellular vesicles in the sample using one or more extracellular vesiclespecific markers.

[0055] Embodiment 42 is the method of any one of embodiments 39-41, wherein the isolating comprises capturing extracellular vesicles in the sample using one or more binding agents specific for one or more extracellular vesicle-specific markers.

[0056] Embodiment 43 is the method of the immediately preceding embodiment, wherein the one or more binding agents is bound to a solid support.

[0057] Embodiment 44 is the method of embodiment 42 or embodiment 43, wherein the binding agent comprises an antibody, a knottin, an aptamer, an affimer, an avimer, a nanobody, a DARPin, a monobody, or an affinity clamp.

[0058] Embodiment 45 is the method of any one of embodiments 42-44, wherein the binding agent comprises an antibody.

[0059] Embodiment 46 is the method of any one of embodiments 42-45, wherein the one or more extracellular vesicle-specific markers comprises one or more of CD81, CD63, CD9, ALIX, CD40, Hsp60, TSG1, or phosphatidylserine.

[0060] Embodiment 47 is the method of any one of embodiments 39-46, wherein the isolating occurs prior to the contacting.

[0061] Embodiment 48 is the method of any one of embodiments 39-47, wherein the isolating occurs prior to the partitioning.

[0062] Embodiment 49 is the method of any one of embodiments 39-48, wherein the isolating occurs after the contacting and prior to the partitioning.

[0063] Embodiment 50 is the method of any one of the preceding embodiments, comprising detecting one or more extracellular vesicles.

[0064] Embodiment 51 is the method of the immediately preceding embodiment, wherein detecting one or more extracellular vesicles comprises:Atty. Docket No. GH0250WO a) contacting the sample with at least one binding agent-oligonucleotide conjugate comprising a binding agent specific for at least one extracellular vesicle-specific marker; and b) before or after the partitioning, detecting a presence, absence, or level of the at least one extracellular vesicle-specific marker by amplifying and / or sequencing an oligonucleotide of the at least one binding agent-oligonucleotide conjugate comprising a binding agent specific for at least one extracellular vesicle-specific marker;thereby detecting the one or more extracellular vesicles in the sample.

[0065] Embodiment 52 is the method of the immediately preceding embodiment, wherein the binding agent comprises an antibody, a knottin, an aptamer, an affimer, an avimer, a nanobody, a DARPin, a monobody, or an affinity clamp.

[0066] Embodiment 53 is the method of embodiment 51 or embodiment 52, wherein the binding agent comprises an antibody.

[0067] Embodiment 54 is the method of any one of embodiments 51-53, wherein the detecting the presence, absence, or level of the at least one extracellular vesicle-specific marker occurs after the partitioning and simultaneously with the detecting the presence, absence, or level of each of the plurality of EV-associated target molecules.

[0068] Embodiment 55 is the method of any one of embodiments 34-52, wherein the at least one extracellular vesicle-specific marker comprises CD81, CD63, CD9, ALIX, CD40, Hsp60, TSG1, or phosphatidylserine.

[0069] Embodiment 56 is the method of any one of embodiments 34-55, wherein the sample is contacted with the at least one binding agent-oligonucleotide conjugate comprising a binding agent specific for at least one extracellular vesicle-specific marker (i) before the sample is contacted with the plurality of binding agent-oligonucleotide conjugates, (ii) at the same time that the sample is contacted with the plurality of binding agent-oligonucleotide conjugates, or (iii) after the sample is contacted with the plurality of binding agent-oligonucleotide conjugates and before the partitioning.

[0070] Embodiment 57 is the method of any one of embodiments 39-56, wherein the at least a portion of the extracellular vesicles in the sample are isolated from other components of the sample using a different extracellular vesicle-specific marker than the at least one extracellular vesicle-specific marker used for detecting the one or more extracellular vesicles in the sample.Atty. Docket No. GH0250WO

[0071] Embodiment 58 is the method of any one of the preceding embodiments, comprising separating extracellular vesicles in the sample that are bound to the one or more binding agents from other components of the sample, thereby providing separated bound extracellular vesicles.

[0072] Embodiment 59 is the method of the immediately preceding embodiments, wherein the separating comprises affinity purification, immunoprecipitation, or a pull down assay.

[0073] Embodiment 60 is the method of any one of embodiments 58-59, wherein the separating occurs prior to the partitioning, and wherein at least a portion of the separated bound extracellular vesicles is subsequently partitioned.

[0074] Embodiment 61 is the method of any one of the preceding embodiments, wherein the sample is a urine sample, an ascites sample, or a saliva sample.

[0075] Embodiment 62 is the method of any one of the preceding embodiments, wherein the sample is a blood sample.

[0076] Embodiment 63 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.

[0077] Embodiment 64 is the method of any one of the preceding embodiments, wherein the sample comprises plasma obtained from a blood sample.

[0078] Embodiment 65 is the method of any one of the preceding embodiments, wherein the sample comprises serum.

[0079] Embodiment 66 is the method of any one of the preceding embodiments, wherein the sample is a tissue sample.

[0080] Embodiment 67 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.

[0081] Embodiment 68 is the method of any one of the preceding embodiments, wherein at least one of the plurality of EV-associated target molecules is a cell type marker.

[0082] Embodiment 69 is the method of the immediately preceding embodiment, wherein the cell type marker is a marker for immune cells or solid tissue cells.

[0083] Embodiment 70 is the method of the immediately preceding embodiment, wherein the cell type marker is selected from markers for colon, lung, breast, skin, prostate, stomach, pancreas, and liver cell type markers.Atty. Docket No. GH0250WO

[0084] Embodiment 71 is the method of any one of the preceding embodiments, wherein at least one of the plurality of EV-associated target molecules is associated with a disease or condition.

[0085] Embodiment 72 is the method of the immediately preceding embodiment, wherein the disease or condition is a cancer.

[0086] Embodiment 73 is the method of any one of the preceding embodiments, wherein the plurality of EV-associated target molecules comprises CD147, 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, CD81, and / or Ephrin A2.

[0087] Embodiment 74 is the method of any one of embodiments 15-73, wherein the plurality of EV-associated target nucleic acids comprises miR-23a, miR-1246, miR-21 let-7a, miR-1229, miR-150, miR-223, miR-4732-5p, miR-301a, miR-486-5p, miR-6803-5p, let-7b-3p, miR-139-3p, miR-145-3p, miR-125a-3p, miR-150-5p, IncRNA colorectal neoplasia differentially expressed - h (CRNDE-h), IncRNA breast cancer anti-estrogen resistance 4 (BCAR4), mRNA keratin associated protein 5-4 (KRTAP5-4), mRNA melanoma associated antigen 3 (MAGEA3), IncRNA urothelial cancer associated 1 (UCA1), circRNA homeodomain interacting protein kinase 3 (HIPK3), IncRNA growth arrest specific 5 (GAS5), LNCV6_116109, LNCV6_98390, LNCV6 84003, LNCV6 98602, LNCV l 08266, LNCV6 38772, and / or IncRNA colon cancer associated 2 (CCAT2).

[0088] Embodiment 75 is the method of any one of the preceding embodiments, wherein the sample is obtained from a subject.

[0089] Embodiment 76 is the method of the immediately preceding embodiment, wherein the subject is an animal.Atty. Docket No. GH0250WO

[0090] Embodiment 77 is the method of any one of embodiments 75-76, wherein the subject is a human.

[0091] Embodiment 78 is the method of any one of embodiments 75-77, wherein the subject has or is at risk of having the disease or condition.

[0092] Embodiment 79 is the method of any one of embodiments 75-78, wherein the method comprises analyzing the plurality of EV-associated target molecules in a subsample of the sample or in a second sample obtained from the same subject from which the first sample is obtained.

[0093] Embodiment 80 is the method of any one of embodiments 75-79, comprising determining a likelihood that the subject has precancer.

[0094] Embodiment 81 is the method of any one of embodiments 75-80, comprising determining a likelihood that the subject has cancer.

[0095] Embodiment 82 is the method of any one of the preceding embodiments, wherein the sequencing comprises generating a plurality of sequencing reads, and wherein the method further comprises mapping the plurality of sequence reads to one or more reference sequences to generate mapped sequence reads, and processing the mapped sequence reads to determine the likelihood that the subject has cancer or precancer.

[0096] Embodiment 83 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.

[0097] Embodiment 84 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.

[0098] Embodiment 85 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 aAtty. Docket No. GH0250WO candidate for a subsequent cancer treatment when the cancer recurrence score is below the cancer recurrence threshold.

[0099] In some embodiments, the results of the methods disclosed herein are used as an input to generate a report. The report may be in a paper or electronic format. For example, true copy number variation, as obtained by the methods disclosed herein, or information derived therefrom, can be displayed directly in such a report. Alternatively or additionally, diagnostic information or therapeutic recommendations which are at least in part based on the methods disclosed herein can be included in the report.

[0100] The various steps of the methods disclosed herein may be carried out at the same or different times, in the same or different geographical locations, e.g. countries, and / or by the same or different people.

[0101] Additional advantages will be set forth in part in the description which follows or may be learned by practice. The advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0102] FIG. 1A illustrates an exemplary workflow according to certain embodiments of the disclosure, beginning with partitioning extracellular vesicles in a sample into a plurality of compartments, e g., wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments. For at least a portion of the partitioned extracellular vesicles, a presence, absence, or level of each of a plurality of EV-associated target molecules is detected using nucleic acid sequencing. Optionally, the sample is contacted with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule. Further optionally, the contacting step can occur prior to the partitioning. Further optionally, the sample may be divided into at least a first subsample and a second subsample (e.g., to be retained as a backup or for use in other analyses), such as prior to the partitioning or prior to the contacting.

[0103] FIG. IB illustrates an exemplary workflow according to certain embodiments of the disclosure, beginning with contacting a sample with a plurality of binding agent-oligonucleotideAtty. Docket No. GH0250WO conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule, (iii) a third binding agent-oligonucleotide conjugate comprising a third oligonucleotide and a third binding agent specific for a third EV-associated target molecule, and (iv) a fourth binding agent-oligonucleotide conjugate comprising a fourth oligonucleotide and a fourth binding agent specific for a fourth EV-associated target molecule. EVs in the sample are then partitioned into a plurality of compartments, e.g., wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments. For at least a portion of the partitioned extracellular vesicles, a presence, absence, or level of each of the plurality of EV-associated target molecules is then detected by amplifying and / or sequencing oligonucleotides of the plurality of binding agent-oligonucleotide conjugates. Optionally, the sample may be divided into at least a first subsample and a second subsample (e.g., to be retained as a backup or for use in other analyses), such as prior to the contacting.

[0104] FIG. 1C illustrates an exemplary workflow according to certain embodiments of the disclosure, beginning with partitioning extracellular vesicles in a sample into a plurality of compartments, e.g., wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments. For at least a portion of the partitioned extracellular vesicles, a presence, absence, or level of each of a plurality of EV-associated target molecules that comprise a plurality of EV-associated nucleic acids is detected using nucleic acid amplification and / or sequencing. Optionally, the sample is contacted with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule. Further optionally, the contacting step can occur prior to the partitioning. Further optionally, the sample may be divided into at least a first subsample and a second subsample (e.g., to be retained as a backup or for use in other analyses), such as prior to the partitioning or prior to the contacting.

[0105] FIG. ID illustrates an exemplary workflow according to certain embodiments of the disclosure, beginning with contacting a sample with a plurality of binding agent-oligonucleotideAtty. Docket No. GH0250WO conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule. The first oligonucleotide comprises a subsequence complementary to a first portion of the second oligonucleotide. The first and second oligonucleotides are then contacted with a polymerase, wherein first and second oligonucleotides of binding agent-oligonucleotide conjugates that are bound to EV-associated target molecules of the same extracellular vesicle undergo extension to form extended oligonucleotides. EVs in the sample are then partitioned into a plurality of compartments, e.g., wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments. For at least a portion of the partitioned extracellular vesicles, a presence, absence, or level of each of the plurality of EV-associated target molecules is detected using amplification of the extended oligonucleotides. Optionally, the sample may be divided into at least a first subsample and a second subsample (e.g., to be retained as a backup or for use in other analyses), such as prior to the contacting.

[0106] FIG. IE illustrates an exemplary workflow according to certain embodiments of the disclosure, beginning with contacting a sample with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule. The first oligonucleotide comprises a subsequence complementary to a first portion of the second oligonucleotide. EVs in the sample are then partitioned into a plurality of compartments, e.g., wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments. The first and second oligonucleotides are then contacted with a polymerase, wherein first and second oligonucleotides of binding agent-oligonucleotide conjugates that are bound to EV-associated target molecules of the same extracellular vesicle undergo extension to form extended oligonucleotides. For at least a portion of the partitioned extracellular vesicles, a presence, absence, or level of each of the plurality of EV-associated target molecules is detected using amplification of the extended oligonucleotides. Optionally, the sample may be divided into at least a first subsample and a second subsample (e.g., to be retained as a backup or for use in other analyses), such as prior to the contacting.Atty. Docket No. GH0250WO

[0107] FIG. IF illustrates an exemplary workflow according to certain embodiments of the disclosure, beginning with contacting a sample with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule. The first oligonucleotide comprises a first subsequence complementary to a first portion of a connector oligonucleotide and the second oligonucleotide comprises a second subsequence complementary to a second portion of the connector oligonucleotide. The first and second oligonucleotides are then contacted with a ligase in the presence of the connector oligonucleotides, wherein first and second oligonucleotides of binding agent-oligonucleotide conjugates that are bound to EV-associated target molecules of the same extracellular vesicle undergo ligation to form ligated oligonucleotides. EVs in the sample are then partitioned into a plurality of compartments, e g., wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments. For at least a portion of the partitioned extracellular vesicles, a presence, absence, or level of each of the plurality of EV-associated target molecules is detected using amplification of the ligated oligonucleotides. Optionally, the sample may be divided into at least a first subsample and a second subsample (e g., to be retained as a backup or for use in other analyses), such as prior to the contacting.

[0108] FIG. 1G illustrates an exemplary workflow according to certain embodiments of the disclosure, beginning with contacting a sample with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule. The first oligonucleotide comprises a first subsequence complementary to a first portion of a connector oligonucleotide and the second oligonucleotide comprises a second subsequence complementary to a second portion of the connector oligonucleotide. EVs in the sample are then partitioned into a plurality of compartments, e.g., wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments. The first and second oligonucleotides are then contacted with a ligase in the presence of the connector oligonucleotides, wherein first and second oligonucleotides of binding agent-oligonucleotideAtty. Docket No. GH0250WOconjugates that are bound to EV-associated target molecules of the same extracellular vesicle undergo ligation to form ligated oligonucleotides. For at least a portion of the partitioned extracellular vesicles, a presence, absence, or level of each of the plurality of EV-associated target molecules is detected using amplification of the ligated oligonucleotides. Optionally, the sample may be divided into at least a first subsample and a second subsample (e.g., to be retained as a backup or for use in other analyses), such as prior to the contacting.

[0109] FIG. 2 is a schematic diagram of an example of a system suitable for use with some embodiments of the disclosure.DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0110] Reference will now be made in detail to certain embodiments of the disclosure. While the disclosure will be described in conjunction with such embodiments, it will be understood that they are not intended to limit the disclosure to those embodiments. On the contrary, the disclosure is intended to cover all alternatives, modifications, and equivalents, which may be included within the disclosure as defined by the appended claims.

[0111] 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.

[0112] 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.

[0113] 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’ orAtty. Docket No. GH0250WO “comprising” the recited components (this interchangeability does not apply to the use of these terms in the claims).

[0114] 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.

[0115] All patents, patent applications, websites, other publications or documents and the like cited herein whether supra or infra, are expressly incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant, unless otherwise indicated.I. Definitions

[0116] As used herein, “primer-annealed DNA or RNA” means DNA or RNA to which at least one primer is annealed.

[0117] 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 resulting 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.

[0118] 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.

[0119] As used herein, “partitioning” refers to physically separating or fractionating extracellular vesicles in a sample into individual compartments. In some embodiments, the plurality ofAtty. Docket No. GH0250WOcompartments are microfluidic chips and / or discs, microarrays, or droplets (such as microdroplets) or droplet crystals (e.g., based on oil-water emulsions), or quantitative PCR-like plates (e.g., nanoplates). In particular embodiments, the plurality of compartments is a plurality of droplets.

[0120] 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. Thus, 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.

[0121] 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.

[0122] As used herein, a “tag” is a molecule, such as a nucleic acid, label, fluorophore, or peptide, 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 purification tag, and / or a detectable tag or label.

[0123] As used herein, a DNA “structural variation” is a mutation comprising a DNA sequence not present in the wild-type genome other than a point mutation (e.g., in which at least 5, 10, 20, or 50 contiguous nucleotides are different relative to the wild type sequence at the corresponding locus). Examples of DNA structural variations include rearrangements, such as translocations, insertions, deletions, duplications, copy -number variants, and inversions. As used herein, a DNA “rearrangement” is a structural variation, wherein the DNA sequence comprises two adjacent sequence portions that are not adjacent to each other in the germline genomic DNA. In some embodiments, a rearrangement is a translocation, gene fusion, insertion, deletion, or inversion. Exemplary rearrangements include products of a translocation, gene fusion, and VDJ recombination. A molecule comprising a structural variation may be referred to as a structuralAtty. Docket No. GH0250WO variant. In some embodiments, an insertion is an insertion of at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides. In some embodiments, a deletion affects sequence spanning the end of the target region, so as to result in a primer being unblocked and undergoing extension in a method described herein.

[0124] “Specifically binds” in the context of a binding agent-oligonucleotide conjugate and an EV-associated target molecule means that under appropriate conditions, the binding agent (such as an antibod) binds the EV-associated target molecule, while at the same time binding of the binding agent to other EV-associated target molecules is minimized. Thus, a particular binding agent binds its target EV-associated molecule to a sufficiently greater extent than to a non-target EV-associated molecule, such as to enable capture or detection of the binding agent-bound EV-associated target molecule. Appropriate binding agent binding conditions are well-known in the art, or can be determined by using routine testing methods.

[0125] A molecule (such as a nucleic acid or a protein) is “derived from cancerous cells” if it originated from a tumor cell. 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).

[0126] 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.

[0127] 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.

[0128] As used herein, “next-generation sequencing” or “NGS” refers to sequencing technologies having increased throughput as compared to traditional Sanger- and capillary electrophoresis-based 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 ofAtty. Docket No. GH0250WO commercially available instruments for performing next-generation sequencing include, but are not limited to, NextSeq, HiSeq, NovaSeq, MiSeq, Ion PGM and Ion GeneStudio S5.

[0129] 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 semi-random 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 single-stranded, 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. Nucleic 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 aAtty. Docket No. GH0250WO10%, 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.

[0130] 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.

[0131] 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’ - 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.

[0132] 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.

[0133] 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.

[0134] 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 beAtty. Docket No. GH0250WOan 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.

[0135] As used herein, “sample” means anything capable of being analyzed by the methods and / or systems disclosed herein.

[0136] 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, electron microscopy-based sequencing, panel sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, wholegenome 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, realtime sequencing, reverse-terminator 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.

[0137] 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.

[0138] 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.

[0139] 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),Atty. Docket No. GH0250WO 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.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 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,Atty. Docket No. GH0250WO BCA, BAC, 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, CABABB, 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.

[0144] “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 fraction 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] “ 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,Atty. Docket No. GH0250WOpancreas, bladder, kidney, and liver. Solid tissue may be normal, precancerous, or cancerous (e.g., a malignant solid tumor such as a carcinoma or sarcoma).

[0149] “ Solid tissue cells” as used herein means cells, respectively, in or derived from a solid tissue. Solid tissue cells exclude circulating cell types, such as cells normally present in blood or lymph.

[0150] 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.

[0151] 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.

[0152] ‘ ‘Extracellular vesicle” as used herein refers to a membrane bound, enclosed object of subcellular size containing cytoplasm and / or other cellular components, e.g., an apoptotic body or exosome, which may have been released from a cell, e.g., a living, apoptotic, or necrotic cell. In some embodiments, an extracellular vesicle has a property (e.g., a component or combination of components, a component protein and / or post-translational modification thereof or combination of proteins and / or modifications, and / or a component nucleic acid (e.g., RNA or DNA) or combination of component nucleic acids) that is specific to one or more cell or tissue types. In some embodiments, a property of an extracellular vesicle is specific to one or more disease or condition types, such as one or more cancer types.

[0153] ‘ ‘Extracellular vesicle-specific 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 EV and is present in greater proportion in EVs than on the outer membrane of intact live cells, cell debris, or in the soluble fraction of a sample. Examples of EV markers include but are not limited to tetraspanines, CD9, CD63, and CD81.

[0154] “Binding agent-oligonucleotide conjugate” as used herein is a molecule comprising a binding agent (such as an antibody, a knottin, an aptamer, an affimer, an avimer, a nanobody, a DARPin, a monobody, or an affinity clamp) and an oligonucleotide. In some embodiments, the oligonucleotide is a DNA. In some embodiments, the binding agent is an antibody.Atty. Docket No. GH0250WO

[0155] “Antibody-oligonucleotide conjugate” as used herein is a chimeric molecule comprising an antibody (such as a monoclonal antibody or an antibody fragment specific for a given EV-associated target molecule) and an oligonucleotide. In some embodiments, the oligonucleotide is aDNA.

[0156] An “extracellular vesicle-associated target molecule” or “EV-associated target molecule” as used herein is a molecule, such as a protein, carbohydrate, lipid, or nucleic acid (such as a DNA or RNA), located in or on an extracellular vesicle, the presence or absence of which can be detected according to methods disclosed herein. The identity of the target molecule need not be known before the detection. The identity of a target molecule may be determined as part of a method described herein, for example, using amplification and / or sequencing.

[0157] “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 determine the presence, absence, or level of one or more components of the sample, such as one or more EV-associated target molecules. Examples of immunoassays may include but are not limited to enzyme-linked immunosorbent assays (ELISAs), sandwich assays, eletrochemiluminescence (ECL) assays, and multiplex assays.

[0158] 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., bi specific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. 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.

[0159] “Or” is used in the inclusive sense, i.e., equivalent to “and / or,” unless the context requires otherwise.II. Exemplary methodsA. Overview

[0160] The present disclosure provides methods and systems for analyzing analyzing a plurality of extracellular vesicle (EV)-associated target molecules in a sample. EVs in biofluids (such asAtty. Docket No. GH0250WOin blood, urine, ascites, or saliva) can comprise biomarkers that have utility for disease detection and characterization. Current methods to characterize biomarkers from single EVs are limited, e.g., in their throughput, dynamic range, multiplexing capability per biomarker type, ability to assay multiple biomarker types, and cost. To address the limitations of current methods, in certain embodiments of the disclosed methods, EV-associated target molecules (such as proteins and / or nucleic acids) in a sample (such as in a biofluid sample) are optionally first contacted with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule. In some embodiments, the EVs in the sample are then partitioned into a plurality of separate reaction partitions (i.e., a plurality of compartments), wherein after the partitioning, only a single EV is present in at least a portion of the plurality of compartments. In some embodiments, as disclosed in further detail elsewhere herein, the partitioning comprises generating a plurality of droplets comprising EVs, wherein only a single EV is present in at least a portion of the plurality of droplets. In some embodiments, the oligonucleotides of the binding agent-oligonucleotide conjugates and / or endogenous EV-associated nucleic acids are then detected using molecular biology techniques (such as amplification and / or sequencing). In some embodiments, as disclosed in further detail elsewhere herein, the detection yields a digital PCR readout. In particular embodiments, the binding agent comprises an antibody.

[0161] Without wishing to be bound by any particular theory, in an individual with cancer, proliferating or activated immune cells and / or dying cancer cells may shed or release more extracellular vesicles into the bloodstream than 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 EV-associated target molecules may change upon carcinogenesis. Variations in presence, absence, and / or level of a plurality of EV-associated target molecules (such as EV-associated proteins and / or nucleic acids) can be an indicator of disease. Thus, the presence, absence, and / or levels of EVs originating from certain cell or tissue types 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.Atty. Docket No. GH0250WO

[0162] In some embodiments, for example, as illustrated in FIG. 1A, methods disclosed herein of analyzing a plurality of extracellular vesicle (EV)-associated target molecules in a sample include steps of (a) partitioning extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles; and (b) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules using nucleic acid sequencing. In some embodiments, the sample is optionally contacted with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule. In some embodiments comprising the step of contacting the sample with a plurality of binding agent-oligonucleotide conjugates, the contacting occurs prior to the partitioning. In some embodiments, the binding agent comprises an antibody.

[0163] In other embodiments, such as illustrated in FIG. IB, methods disclosed herein comprise (a) contacting the sample with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule, (iii) a third binding agent-oligonucleotide conjugate comprising a third oligonucleotide and a third binding agent specific for a third EV-associated target molecule, and (iv) a fourth binding agent-oligonucleotide conjugate comprising a fourth oligonucleotide and a fourth binding agent specific for a fourth EV-associated target molecule; (b) partitioning the extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles; and (c) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules by amplifying and / or sequencing oligonucleotides of the plurality of binding agent-oligonucleotide conjugates. In some embodiments, the binding agent comprises an antibody.Atty. Docket No. GH0250WO

[0164] In some embodiments of the disclosed methods, such as illustrated in FIG. 1C, a method of analyzing a plurality of EV-associated target molecules in a sample comprises (a) partitioning the extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles; and (b) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules, wherein the plurality of EV-associated target molecules comprise a plurality of EV-associated nucleic acids, wherein the detecting comprises nucleic acid amplification and / or sequencing. In some embodiments, the sample is optionally contacted with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule. In some embodiments comprising the step of contacting the sample with a plurality of binding agent-oligonucleotide conjugates, the contacting occurs prior to the partitioning. In some embodiments, the binding agent comprises an antibody.

[0165] The detection of a presence, absence, or level of each of the plurality of EV-associated target molecules may be performed in a manner that renders the detection dependent on the proximity of (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule, such as a proximity ligation assay or proximity extension assay. A workflow including a proximity extension assay is illustrated in FIGS. ID and IE, in which following extension, a further amplification may be performed in which barcodes are added to the oligonucleotides. A workflow including a proximity ligation assay is illustrated in FIGS. IF and 1G, in which following ligation, a further amplification may be performed in which barcodes are added to the oligonucleotide labels.

[0166] In some embodiments, for example, as illustrated in FIGS. ID and IE, the method comprises (a) contacting the sample with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, andAtty. Docket No. GH0250WO (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule; wherein the first oligonucleotide comprises a subsequence complementary to a first portion of the second oligonucleotide; (b) partitioning extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles; (c) before or after the partitioning, contacting the first and second oligonucleotides with a polymerase, wherein first and second oligonucleotides of binding agent-oligonucleotide conjugates that are bound to EV-associated target molecules of the same extracellular vesicle undergo extension to form extended oligonucleotides; and (d) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules using amplification of the extended oligonucleotides. In some embodiments the first and second oligonucleotides are contacted with the polymerase before the partitioning and optionally after the contacting the sample with a plurality of binding agent-oligonucleotide conjugates (FIG. ID). In some embodiments the first and second oligonucleotides are contacted with the polymerase after the partitioning (FIG. IE). In some embodiments, the binding agent comprises an antibody.

[0167] In other embodiments, such as illustrated in FIGS. IF and 1G, the method comprises (a) contacting the sample with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule; wherein the first oligonucleotide comprises a first subsequence complementary to a first portion of a connector oligonucleotide and the second oligonucleotide comprises a second subsequence complementary to a second portion of the connector oligonucleotide; (b) partitioning extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles; (c) before or after the partitioning, contacting the first and second oligonucleotides with a ligase in the presence of the connector oligonucleotides, wherein first and second oligonucleotides of binding agent-oligonucleotide conjugates that are bound to EV-associated target molecules of the same extracellular vesicle undergo ligation to form ligatedAtty. Docket No. GH0250WOoligonucleotides; and (d) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules using amplification of the ligated oligonucleotides. In some embodiments the first and second oligonucleotides are contacted with the ligase before the partitioning and optionally after the contacting the sample with a plurality of binding agent-oligonucleotide conjugates (FIG. IF). In some embodiments the first and second oligonucleotides are contacted with the ligase after the partitioning (FIG. 1G). In some embodiments, the binding agent comprises an antibody.

[0168] In some embodiments, the plurality of binding agent-oligonucleotide conjugates further comprises at least a third binding agent-oligonucleotide conjugate comprising a third binding agent specific for a third EV-associated target molecule. In certain embodiments, the plurality of binding agent-oligonucleotide conjugates further comprises at least a fourth binding agent-oligonucleotide conjugate comprising a fourth binding agent specific for a fourth EV-associated target molecule. In some embodiments, the plurality of binding agent-oligonucleotide conjugates further comprises at least a fifth binding agent-oligonucleotide conjugate comprising a fifth binding agent specific for a fifth EV-associated target molecule.

[0169] In some embodiments of the disclosed methods, the plurality of binding agent-oligonucleotide conjugates comprises 2-30, such as 2-25, 2-20, 2-15, 2-10, 2-5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more than 30 binding agent-oligonucleotide conjugates. In some embodiments, the plurality of EV-associated target molecules comprises 2-30, such as 2-25, 2-20, 2-15, 2-10, 2-5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more than 30 EV-associated target molecules.

[0170] In some embodiments of the disclosed methods, the binding agent comprises an antibody, a knottin, an aptamer, an affimer, an avimer, a nanobody, a DARPin, a monobody, or an affinity clamp. In particular embodiments, the binding agent comprises an antibody.

[0171] In certain embodiments, the plurality of EV-associated target molecules comprises one or more proteins. In some embodiments, the one or more proteins comprise one or more extracellular vesicle surface proteins.

[0172] In some embodiments, the plurality of EV-associated target molecules comprises one or more EV-associated target nucleic acids. In certain embodiments, the plurality of EV-associated target nucleic acids comprises one or more DNAs. In certain embodiments, the plurality of EV-associated target nucleic acids comprises one or more RNAs. In some embodiments, the one or more RNAs comprise one or more messenger RNAs (mRNAs), one or more ribosomal RNAsAtty. Docket No. GH0250WO (rRNAs), one or more transfer RNAs (tRNAs), one or more microRNAs (miRNAs), one or more small nuclear RNAs (snRNAs), one or more small interfering RNAs (siRNAs), and / or one or more long non-coding RNAs (IncRNAs). In some embodiments, the one or more RNAs comprise one or more mRNAs. In some embodiments, the one or more RNAs comprise one or more rRNAs. In some embodiments, the one or more RNAs comprise one or more tRNAs. In some embodiments, the one or more RNAs comprise one or more miRNAs. In some embodiments, the one or more RNAs comprise one or more snRNAs. In some embodiments, the one or more RNAs comprise one or more siRNAs. In some embodiments, the one or more RNAs comprise one or more IncRNAs.

[0173] In some embodiments, at least one of the plurality of EV-associated target molecules is a cell type marker. In particular embodiments, the cell type marker is a marker for immune cells or solid tissue cells. In some embodiments, the cell type marker is selected from markers for colon, lung, breast, skin, prostate, stomach, pancreas, and liver cell type markers.

[0174] In some embodiments, at least one of the plurality of EV-associated target molecules is associated with a disease or condition. In particular embodiments, the disease or condition is a cancer.

[0175] In some embodiments, the plurality of EV-associated target molecules comprises CD147, 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 [3-1 (TGF|3-1), melanoma antigen 3 / 6 (MAGE-3 / 6), CD37, CD53, CD81, CD81, and / or Ephrin A2.

[0176] In some embodiments, the plurality of EV-associated target nucleic acids comprises miR-23a, miR-1246, miR-21 let-7a, miR-1229, miR-150, miR-223, miR-4732-5p, miR-301a, miR-Atty. Docket No. GH0250WO 486-5p, miR-6803-5p, let-7b-3p, miR-139-3p, miR-145-3p, miR-125a-3p, miR-150-5p, IncRNA colorectal neoplasia differentially expressed - h (CRNDE-h), IncRNA breast cancer anti-estrogen resistance 4 (BCAR4), mRNA keratin associated protein 5-4 (KRTAP5-4), mRNA melanoma associated antigen 3 (MAGEA3), IncRNA urothelial cancer associated 1 (UCA1), circRNA homeodomain interacting protein kinase 3 (HIPK3), IncRNA growth arrest specific 5 (GAS5), LNCV6_116109, LNCV6_98390, LNCV6_84003, LNCV6_98602, LNCV_108266, LNCV6_38772, and / or IncRNA colon cancer associated 2 (CCAT2).

[0177] In some embodiments, the method comprises analyzing the plurality of EV-associated target molecules in a sub sample of the sample or in a second sample obtained from the same subject from which the first sample is obtained.

[0178] In some embodiments, the sample is obtained from a subject, optionally wherein the subject has or is at risk of having a disease or condition. In some embodiments, the disease or condition is a cancer or precancer, and certain embodiments of the disclosed methods comprise determining the likelihood that the subject has the disease or condition, such as the cancer or precancer. 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, optionally wherein the sample is obtained at one or more preselected time points following the one or more previous cancer treatments.

[0179] In particular embodiments, the method comprises determining a likelihood that the subject has precancer. In particular embodiments, the method comprises determining a likelihood that the subject has cancer.

[0180] In some embodiments, the sequencing comprises generating a plurality of sequencing reads, and wherein the method further comprises mapping the plurality of sequence reads to one or more reference sequences to generate mapped sequence reads, and processing the mapped sequence reads to determine the likelihood that the subject has cancer or precancer.

[0181] 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, optionally wherein the sample is obtained at one or more preselected time points following the one or more previous cancer treatments. Some embodiments of the disclosed methods further comprise 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 theAtty. Docket No. GH0250WO subject is determined to be at lower risk for cancer recurrence when the cancer recurrence score is below the predetermined threshold. Some embodiments further comprise 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.

[0182] 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 a 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.B. Partitioning

[0183] In some instances, extracellular vesicles (EVs) in asample are partitioned into a plurality of compartments. After the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles. In some embodiments, the plurality of compartments are microfluidic chips and / or discs, microarrays, or droplets (such as microdroplets) or droplet crystals (e.g., based on oil-water emulsions), or quantitative PCR-like plates (e.g., nanoplates). In particular embodiments, the plurality of compartments is a plurality of droplets.

[0184] In some embodiments, the plurality of compartments is a plurality of droplets, and a detection step comprises digital PCR, such as droplet digital PCR. A droplet digital PCRAtty. Docket No. GH0250WO(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.

[0185] The digital droplet PCR reaction in a ddPCR workflow may be prepared in a tube. The ddPCR mix is then partitioned into individual droplets using a droplet generator. The emulsion is collected, e.g., in a vial, and PCR is performed. A digital PCR instrument can separately amplify nucleic acids present in the individual droplets, such as oligonucleotides of binding agent-oligonucleotide conjugates (such as of 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 etal., 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.

[0186] In some embodiments, the plurality of compartments is a plurality of droplets, and the detection step comprises sequencing (such as using next generation sequencing) oligonucleotides of the plurality of 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 from each partitioned compartment (e.g., each droplet) 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 moleculeds comprising nucleic acids in each partition. Analyzing EV-associated DNA may comprise detecting or quantifying DNA of interestAtty. Docket No. GH0250WO 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.

[0187] In some embodiments, a first subsample of the sample is partitioned as described herein, and a second subsample, and optionally a third subsample are not partitioned and are retained as backups and / or for use in other analyses. Accordingly, the disclosed methods can be combined with analysis of one or more additional biomarkers, such as analysis of one or more additional biomarkers using the second subsample and / or the third subsample (or one or more additional subsamples). 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 a 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.C. Adapter ligation or addition; tagging

[0188] In some embodiments, the methods comprise ligating adapters to one or more EV-associated nucleic acids, such as RNA or DNA. In some embodiments, the ligating adapters to the one or more EV-associated nucleic acids produces adapter-ligated EV-associated nucleic acids, such as adapter-ligated, EV-associated DNA or adapter-ligated, EV-associated RNA. In some embodiments, DNA molecules can be subjected to blunt-end ligation with blunt-ended adapters. In some embodiments, DNA molecules can be subjected to sticky-end ligation with sticky-ended adapters. Nucleic acid molecules can be ligated to adapters at either one end or both ends. DNA molecules can be ligated with at least partially double stranded adapter (e.g., a Y shaped or bell-shaped adapter).Atty. Docket No. GH0250WO

[0189] In some embodiments, the ligation step can take place prior to amplifying or sequencing the EV-associated nucleic acids. In some embodiments, the ligation step can take place prior to or after partitioning the EVs into a plurality of compartments. In some embodiments, the ligation step can take place prior to partitioning the EVs into a plurality of compartments. In some embodiments, the ligation step can take place prior to or after EVs into a plurality of compartments and prior to or after sequencing the EV-associated nucleic acids. In some embodiments, the ligation step can take place after EVs into a plurality of compartments and prior to the sequencing the EV-associated nucleic acids.

[0190] A ligase and adapters are added to ligate EV-associated nucleic acids in the sample with an adapter on one or both ends, i.e. to form adapted DNA or RNA. As used herein, “adapter” refers to short nucleic acids (e.g., less than about 500, less than about 100 or less than about 50 nucleotides in length, or be 20-30, 20-40, 30-50, 30-60, 40-60, 40-70, 50-60, 50-70, 20-500, or 30-100 bases from end to end). In some embodiments, the adapters are at least partially doublestranded and can be ligated to the end of a given sample EV-associated DNA molecule. In some instances, two adapters can be ligated to a single sample EV-associated DNA molecule, with one adapter ligated to each end of the nucleic acid molecule.

[0191] In some embodiments, a DNA ligase used in ligation reactions can act on both single strand DNA nicks and double stranded DNA ends. In some cases, the ligase is T4 DNA ligase or T3 DNA ligase. Adapters can include nucleic acid primer binding sites to permit amplification of an EV-associated DNA molecule flanked by adapters at both ends, and / or a sequencing primer binding site, including primer binding sites for sequencing applications, such as various next generation sequencing (NGS) applications. Adapters can include a sequence for hybridizing to a solid support, e.g., a flow cell sequence. Adapters can also include binding sites for capture probes, such as an oligonucleotide attached to a flow cell support or the like. Adapters can also include sample indexes and / or molecular barcodes. These are typically positioned relative to amplification primer and sequencing primer binding sites, such that the sample index and / or molecular barcode is included in amplicons and sequencing reads of a given EV-associated DNA molecule. Adapters of the same or different sequence can be linked to the respective ends of an EV-associated DNA molecule. In some cases, adapters of the same or different sequence are linked to the respective ends of the EV-associated DNA molecule except that the sample index and / or molecular barcode differs in its sequence. In some embodiments, the adapter is an asymmetric adapter, such as a Y-shaped adapter in which one end is blunt ended or tailed asAtty. Docket No. GH0250WO described herein, for joining to a nucleic acid molecule, which is also blunt ended or tailed with one or more complementary nucleotides to those in the tail of the adapter. In another exemplary embodiment, an adapter is a bell-shaped adapter that includes a blunt or tailed end for joining to an EV-associated DNA molecule to be analyzed. Other exemplary adapters include T-tailed, C-tailed or hairpin shaped adapters and bubble adapters. For example, a 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. Adapters used in the methods of the present disclosure can comprise one or more known modified nucleosides, such as methylated nucleosides.

[0192] In instances where two adapters are ligated to a sample nucleic acid (one at each end), either or both of the adapters may comprise one or more known modified nucleosides. Where a conversion procedure is used, typically, the primer binding site(s), sequencing primer binding site(s), sample index(es) and / or molecular barcode(s), if present, do not comprise the known modified nucleosides that change base pairing specificity as a result of the conversion procedure.

[0193] In some embodiments, adapters may be added to the EV-associated nucleic acids or a subsample thereof. Adapters can be ligated to EV-associated nucleic acids at any point in the methods herein. In some embodiments, adapters are ligated to the EV-associated nucleic acids in a sample. In some such embodiments, adapters are ligated to EV-associated nucleic acids after a step of partitioning as disclosed herein. In some such embodiments, the adapter-ligated EV-associated nucleic acids is amplified prior to a step of sequencing.

[0194] In some embodiments, the disclosed methods comprise analyzing EV-associated nucleic acids in a sample. In such methods, adapters may be added to the EV-associated nucleic acids. 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), before, or after an amplification step. In some embodiments, adapters are added by other approaches, such as ligation. In some such methods, first adapters are added to the 3’ ends of the nucleic acids by ligation, which may include ligation to single-stranded EV-associated nucleic acids. In some embodiments, prior to any partitioning or capturing steps, first adapters are added to the nucleic acids by ligation, which may include ligation to single-stranded EV-associated nucleic acids (e.g., to the 3’ ends thereof). In some embodiments, the capture probes can beAtty. Docket No. GH0250WO isolated after partitioning and ligation. For example, EV-associated nucleic acids in a partitioned compartment can be ligated with adapters. Adapters 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. 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.

[0195] 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 singlestranded. 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 single-stranded and coated with SSB, is placed in a phosphorylation / ligation dual reaction with directional dsDNANGS adapters that contain singlestranded 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 the 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.

[0196] 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 nucleotidesAtty. Docket No. GH0250WOof 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.

[0197] In some embodiments, 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.

[0198] 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.

[0199] In some embodiments, the EV-associated nucleic acids are linked at both ends to Y-shaped adapters including primer binding sites and tags. The molecules are amplified.D. Molecular Tagging

[0200] In some embodiments, oligonucleotides of the plurality of binding agent-oligonucleotide conjugates (such as antibody-oligonucleotide conjugates) and / or EV-associated nucleic acids (such as adapted EV-associated nucleic acids) may be tagged with sample indexes and / or molecular barcodes (referred to generally as “tags”). In some embodiments, EV-associated nucleic acids are tagged, e.g., with barcodes. In some embodiments, the oligonucleotides and / or EV-associated nucleic acids molecules of the sample comprise barcodes. 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, oligonucleotides and / or EV-associated nucleic acids molecules can bear a sample tag or sample index (which distinguishes molecules in one sample from those in a different sample), and / or a molecular tag / molecular barcode (which distinguishes different molecules, such as different EV-associated target molecules bound to different binding agent-oligonucleotide conjugates (such as antibody-oligonucleotide conjugates), from one another (in both unique and non-unique tagging scenarios)).

[0201] 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 thatAtty. Docket No. GH0250WO 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.

[0202] 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, molecular tags, or molecular indexes 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.

[0203] For example, barcodes can be used to allow the origin of the EV-associated nucleic acids (e.g., the subject, biological sample (e.g., samples collected at various time points), and / or the individual EV partitioned into a compartment, to be identified, e.g., following pooling of a plurality of samples for parallel sequencing. Tags comprising barcodes can be incorporated into or otherwise joined to adapters. Tags can be incorporated by ligation, overlap extension PCR among other methods. 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 of the disclosure 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 specific partition. In embodiments employing multiple different tags to label a specific partition, the set of tags used to label one partition canAtty. Docket No. GH0250WObe 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 e0146638 (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).

[0204] Tags may be incorporated into or otherwise joined to adapters by chemical synthesis, ligation (e.g., as described above, e.g. by blunt-end ligation or sticky-end ligation), or overlap extension polymerase chain reaction (PCR), among other methods. Such adapters are ultimately joined to the oligonucleotide or EV-associated nucleic acid. In other embodiments, one or more rounds of amplification cycles (e.g., PCR amplification) may be applied to introduce sample indexes to a nucleic acid molecule using conventional nucleic acid amplification methods. The amplifications may be conducted in one or more reaction mixtures (e.g., a plurality of microwells in an array). Molecular barcodes and / or sample indexes may be introduced simultaneously, or in any sequential order. In some embodiments, molecular barcodes and / or sample indexes are introduced prior to and / or after any conversion procedure. In the case of molecular barcodes and / or sample indexes being introduced through amplification processes, the conversion step, if used, will occur before the molecular barcodes and / or sample indexes are introduced. In some embodiments, sample indexes are incorporated through overlap extension polymerase chain reaction (PCR).

[0205] In some embodiments, the tags may be located at one end or at both ends of the oligonucleotide or EV-associated nucleic acid molecule. In some embodiments, tags are predetermined or random or semi-random sequence oligonucleotides. In some embodiments, the tag(s) may together be less than about 500, 200, 100, 50, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides in length. Typically tags are about 5 to 20 or 6 to 15 nucleotides in length. The tags may be linked to oligonucleotides EV-associated nucleic acids molecules randomly or non-randomly.Atty. Docket No. GH0250WO

[0206] In some embodiments, each sample or partition (discussed below), or each binding agent-oligonucleotide conjugate (such as each antibody-oligonucleotide conjugate) specific for a given EV-associated target molecule, is uniquely tagged with a sample index or a combination of sample indexes. In some embodiments, each nucleic acid molecule of a sample or sub-sample is uniquely tagged with a molecular barcode or a combination of molecular barcodes. In other embodiments, a plurality of molecular barcodes may be used such that molecular barcodes are not necessarily unique to one another in the plurality (e.g., non-unique molecular barcodes). In these embodiments, molecular barcodes are generally attached (e.g., by ligation as part of an adapter) to individual molecules such that the combination of the molecular barcode and the sequence it may be attached to creates a unique sequence that may be individually tracked.Detection of non-unique molecular barcodes in combination with endogenous sequence information (e.g., the beginning (start) and / or end (stop) genomic location / position corresponding to the sequence of the original DNA molecule in the sample, start and stop genomic positions corresponding to the sequence of the original DNA molecule in the sample, the beginning (start) and / or end (stop) genomic location / position of the sequence read that is mapped to the reference sequence, start and stop genomic positions of the sequence read that is mapped to the reference sequence, sub-sequences of sequence reads at one or both ends, length of sequence reads, and / or length of the original DNA molecule in the sample) typically allows for the assignment of a unique identity to a particular molecule. In some embodiments, beginning region comprises the first 1, first 2, the first 5, the first 10, the first 15, the first 20, the first 25, the first 30 or at least the first 30 base positions at the 5' end of the sequencing read that align to the reference sequence. In some embodiments, the end region comprises the last 1, last 2, the last 5, the last 10, the last 15, the last 20, the last 25, the last 30 or at least the last 30 base positions at the 3' end of the sequencing read that align to the reference sequence. The length, or number of base pairs, of an individual sequence read are also optionally used to assign a unique identity to a given 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, and / or a complementary strand.

[0207] 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 oligonucleotides and / or EV-associated nucleic acids of a particular group bear a different tag. It is to be noted that when barcodes are used as tags, and whenAtty. Docket No. GH0250WO 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).

[0208] In some embodiments, molecular barcodes are introduced at an expected ratio of a set of identifiers (e.g., a combination of unique or non-unique molecular barcodes) to oligonucleotides and / or nucleic acid molecules associated with an EV. One example format uses from about 2 to about 1,000,000 different molecular barcode sequences, or from about 5 to about 150 different molecular barcode sequences, or from about 20 to about 50 different molecular barcode sequences, ligated to both ends of an oligonucleotide or EV-associated nucleic acid.Alternatively, from about 25 to about 1,000,000 different molecular barcode sequences may be used. For example, 20-50 x 20-50 molecular barcode sequences (i.e., one of the 20-50 different molecular barcode sequences can be attached to each end of the oligonucleotide or EV-associated nucleic acid) can be used. Such numbers of identifiers are typically sufficient for different molecules having the same start and stop points to have a high probability (e.g., at least 94%, 99.5%, 99.99%, or 99.999%) of receiving different combinations of identifiers. In some embodiments, about 80%, about 90%, about 95%, or about 99% of molecules have the same combinations of molecular barcodes. In some embodiments, about 50 to about 50,000 different tags (e.g., between about 6 and 220 barcode combinations) can suffice to uniquely tag all oligonucleotides or EV-associated nucleic acids.

[0209] In some embodiments, the assignment of unique or non-unique molecular barcodes in reactions is performed using methods and systems described for example, U.S. Patent Application Nos. 20010053519, 20030152490, and 20110160078, and U.S. PatentNos.6,582,908, 7,537,898, 9,598,731, and 9,902,992, each of which is hereby incorporated by reference in its entirety. Alternatively, in some embodiments, different oligonucleotides or EV-associated nucleic acids may be identified using only endogenous sequence information (e.g.,Atty. Docket No. GH0250WOstart and / or stop positions, sub-sequences 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 peer 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, a format uses 20-50 different tags (e.g., barcodes) ligated to both ends of oligonucleotides or EV-associated nucleic acids. For example, 35 different tags (e.g., barcodes) ligated to both ends of oligonucleotides or EV-associated nucleic acids 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.

[0212] 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 oligonucleotides or EV-associated nucleic acids 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 a 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.Atty. Docket No. GH0250WO

[0213] In some embodiments, the method includes adding one or more internal control nucleic acids and forward and reverse primers for amplifying the internal control nucleic acids. The internal control nucleic acids may be added before amplification using the primers that anneal upstream and downstream of the rearrangement breakpoints. The forward and reverse primers for amplifying the internal control nucleic acids may be included with, or added at the same time as, the primers that anneal upstream and downstream of the rearrangement breakpoints. The internal control may comprise or consist of sequences that do not occur in the genome or transcriptome of the subject, or that do not occur in the genome of the species of which the subject is a member (e.g., the human genome). The forward and / or reverse primers for amplifying the internal control nucleic acids may comprise sequences that are not complementary to any sequence in the transcriptome or genome of the subject, e.g., the human transcriptome or genome. The internal control nucleic acids may be used to ensure that the amplification process proceeded as designed. As such, the method may comprise detecting (e.g., sequencing) molecules amplified from and / or captured by the one or more internal control nucleic acids. The method can comprise comparing an amount of internal control nucleic acids (e.g., number of molecules or reads detected that correspond to an internal control nucleic acids sequence) to a predetermined threshold, and either rejecting sequencing results if the predetermined threshold is not met or accepting sequencing results if the predetermined threshold is met. The predetermined threshold may be established, e.g., based on historical data or by testing the method on samples of nucleic acids from test subjects, such as healthy volunteers. For example, amplification and detection of the one or more internal control nucleic acids provides confirmation that the amplification process proceeded properly, thus reducing the likelihood of a false negative.E. Amplification

[0214] In some embodiments, a plurality of EV-associated target nucleic acids (such as DNA or RNA) is amplified. In some embodiments, one or more barcodes are added to the plurality of EV-associated target nucleic acids or amplification products thereof. In certain embodiments, one or more of the oligonucleotides of the plurality of binding agent-oligonucleotide conjugates (such as antibody-oligonucleotide conjugates) is amplified. In some embodiments, the one or more oligonucleotides comprises at least one tag. In some embodiments, the at least one tag comprises a molecular barcode.Atty. Docket No. GH0250WO

[0215] In some embodiments, the amplifying comprises PCR, digital PCR, reverse transcription PCR (RT-PCR), rolling circle amplification, and / or linear amplification. In certain embodiments wherein the plurality of EV-associated target nucleic acids comprise RNA, the amplifying comprises RT-PCR, such as prior to digital PCR (such as droplet digital PCR) or sequencing (such as next generation sequencing). In some embodiments, the detecting comprises amplifying oligonucleotides of the binding agent-oligonucleotide conjugates (such as antibody-oligonucleotide conjugates) that are hybridized to each other (e.g., as part of a proximity extension assay or proximity ligation assay; in the latter case amplifying would follow ligating). In some embodiments, the amplifying is quantitative, e.g., as in qPCR. In some embodiments, the detecting comprises sequencing the amplified oligonucleotides.

[0216] In certain embodiments, the amplifying occurs after a step of partitioning extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles; and prior to a step of detecting a presence, absence, or level of each of a plurality of EV-associated target molecules (such as using digital PCR (such as droplet digital PCR) or sequencing (such as next generation sequencing)) as described elsewhere herein. In some such embodiments, the plurality of compartments comprise droplets. In some such embodiments, EV-associated target nucleic acids and / or one or more of the oligonucleotides of the plurality of binding agent-oligonucleotide conjugates (such as antibody-oligonucleotide conjugates) are amplified after the step of partitioning, and are subsequently amplified.

[0217] In some embodiments, EV-associated target nucleic acids and / or one or more of the oligonucleotides of the plurality of binding agent-oligonucleotide conjugates (such as antibody-oligonucleotide conjugates) are partitioned into a plurality of droplets using a droplet generator (e.g., as part of a droplet digital PCR process), and are subsequently amplified using RT-PCR (e.g., to generate cDNA from EV-associated nucleic acids comprising RNA) and digital PCR. In some embodiments, EV-associated target nucleic acids and / or one or more of the oligonucleotides of the plurality of binding agent-oligonucleotide conjugates (such as antibody-oligonucleotide conjugates) are partitioned into a plurality of droplets using a droplet generator (e.g., as part of a droplet digital PCR process), and are subsequently amplified using digital PCR. In some embodiments, EV-associated target nucleic acids and / or one or more of the oligonucleotides of the plurality of binding agent-oligonucleotide conjugates (such as antibody -Atty. Docket No. GH0250WO oligonucleotide conjugates) are partitioned into a plurality of droplets using a droplet generator, and are subsequently amplified using RT-PCR (e.g., to generate cDNA from EV-associated nucleic acids comprising RNA) and sequenced (such as using next generation sequencing). In some embodiments, EV-associated target nucleic acids and / or one or more of the oligonucleotides of the plurality of binding agent-oligonucleotide conjugates (such as antibody -oligonucleotide conjugates) are partitioned into a plurality of droplets using a droplet generator, and are subsequently sequenced (such as using next generation sequencing).

[0218] In some embodiments, a plurality of EV-associated nucleic acids comprising adapted DNA or adapted cDNA can be amplified (e.g. by PCR) prior to, or as part of, sequencing. In some embodiments, a plurality of EV-associated nucleic acids are amplified as part of a digital PCR reaction. In some embodiments, a plurality of EV-associated nucleic acids comprising RNA is reverse transcribed using RT-PCR and the resultant cDNA is sequenced (such as using next generation sequencing). In some embodiments, a plurality of EV-associated nucleic acids comprising RNA is reverse transcribed using RT-PCR and the resultant cDNA is amplified (e g. by PCR) prior to, or as part of, sequencing(such as using next generation sequencing). In some embodiments, a plurality of EV-associated nucleic acids comprising RNA is reverse transcribed using RT-PCR and the resultant cDNA is amplified using digital PCR (such as droplet digital PCR). In some embodiments, a plurality of EV-associated nucleic acids comprising DNA is amplified prior to a step of subjecting adapter-ligated EV-associated DNA to sequencing. In some embodiments, a plurality of EV-associated nucleic acids comprising DNA is amplified after ligating adapters to the DNA and / or before sequencing the DNA. In some embodiments, a plurality of EV-associated nucleic acids comprising RNA is reverse transcribed using RT-PCR and the resultant cDNA is amplified prior to a step of subjecting adapter-ligated cDNA to sequencing. In some embodiments, a plurality of EV-associated nucleic acids comprising RNA is reverse transcribed using RT-PCR and the resultant cDNA is amplified after ligating adapters to the cDNA and / or before sequencing the cDNA.

[0219] Amplification is typically primed by primers binding to primer binding sites in adapters flanking a DNA molecule to be amplified. Amplification methods can involve cycles of denaturation, annealing and extension, resulting from thermocycling or can be isothermal as in transcription-mediated amplification. For example, sample nucleic acids flanked by adapters can be amplified by PCR and other amplification methods. Amplification methods of use herein can include any suitable methods, such as known to those of ordinary skill in the art. In someAtty. Docket No. GH0250WOembodiments, amplification is primed by primers binding to primer binding sites in adapters flanking a DNA molecule to be amplified. Amplification methods can involve cycles of denaturation, annealing and extension, resulting from thermocycling, such as polymerase chain reaction (PCR), or can be isothermal, such as in linear amplification methods, transcription-mediated amplification, recombinase polymerase amplification (RPA), helicase dependent amplification (HDA), rolling-circle amplification (RCA) (Blanco et al., J. Biol. Chem., 264, 8935-8940, 1989), or hyperbranched rolling circle amplification (Lizard et al., Nat. Genetics, 19, 225-232, 1998). Other amplification methods include the ligase chain reaction, strand displacement amplification, nucleic acid sequence based amplification, and self-sustained sequence based replication.

[0220] In some embodiments, an amplification of the DNA in a sample comprises amplifying rolling-circle amplification (RCA). In some embodiments, RCA comprises circularizing a DNA template (e.g., DNA in the converted sample). In some embodiments, RCA comprises copying the circularized DNA template using a rolling circle polymerase to generate a plurality of circularized DNA templates. In some embodiments, the rolling circle polymerase is a phi29 DNA polymerase. Exemplary methods of RCA are provided, e.g., in Lou etal., Proc. Natl. Acad. Sci. 110 (49) 19872-19877 (2013). In some embodiments, the RCA occurs prior to a step of sequencing the DNA.

[0221] This may be an additional amplification step subsequent to an earlier amplification step, such as amplification as described elsewhere herein. In some embodiments, amplification of adapted DNA comprises RCA, e.g., as described above. In some embodiments, RCA comprises copying the circularized DNA template using a rolling circle polymerase to generate a plurality of circularized DNA templates. In some embodiments, the rolling circle polymerase is a phi29 DNA polymerase.

[0222] In some embodiments, sequencing DNA that was amplified using RCA (e.g., as described elsewhere herein) provides sequence reads comprising multiple copies of the sequence of an original sample molecule or converted molecule and the copies are used to determine a consensus sequence of the original sample molecule or converted molecule.

[0223] In some embodiments, the present methods perform dsDNA ligations with T-tailed and C-tailed adapters. The addition of C-tailed adapters can increase ligation efficiency because the A-tailing reaction can also add G-tails to a small portion of the DNA molecules, when the A tailing is performed in the presence of dGTP, such as when the A-tailing is performed in the same reactionAtty. Docket No. GH0250WOas the end repair. The use of T-tailed and C-tailed adapters can result in amplification of at least 50, 60, 70 or 80% of double stranded nucleic acids before. The present methods can increase the amount or number of amplified molecules relative to control methods performed with T-tailed adapters alone by at least 10, 15 or 20%.

[0224] In some embodiments, adapted EV-associated DNA or adapted EV-associated cDNA (e g., following reverse transcription of EV-associated target molecules comprising RNA) is amplified before sequencing. Amplification may in some cases be before one or more isolation and / or capture steps. In some embodiments, adapters are ligated to the EV-associated DNA or cDNA before or simultaneously with amplification.

[0225] In some embodiments, amplification of the EV-associated DNA (e.g., adapter ligated DNA) comprises using a DNA polymerase. In some embodiments, the DNA polymerase may be Q5® High-Fidelity DNA Polymerase, Q5U® Hot Start High-Fidelity DNA Polymerase, Phusion® High-Fidelity DNA Polymerase, One / aq” DNA Polymerase, Taq DNA Polymerase, LongAmp® Taq DNA Polymerase, Hemo ¥A&aTaq, Epimark® Hot Start Taq DNA Polymerase, Bst DNA Polymerase, Full Length, Bst DNA Polymerase, Large Fragment, Bst 2.0 DNA Polymerase, Bst 3.0 DNA Polymerase, Bsu DNA Polymerase, Large Fragment, phi29 DNA Polymerase, phi29-XT DNA Polymerase, Sulfolobus DNA Polymerase IV, Therminator™ DNA Polymerase, T7 DNA Polymerase, DNA Polymerase I (E. coli), DNA Polymerase I, Large (Klenow) Fragment (“Klenow fragment”), Klenow Fragment (3 '— 5' exo-), T4 DNA Polymerase, Vent® DNA Polymerase, Vent® (exo-) DNA Polymerase, Deep Vent® DNA Polymerase, Deep Vent® (exo-) DNA Polymerase, or any combination thereof.F. Detecting

[0226] Embodiments of the disclosed methods comprises detecting a presence, absence, or level of each of a plurality of EV-associated target molecules. In some examples, detecting a level of each of a plurality of EV-associated target molecules comprises quantifying each of the plurality of EV-associated target molecules, such within and / or on an EV of a partitioned compartment. For example, use of the disclosed methods in particular examples permits detection of oligonucleotides of a plurality of binding agent-oligonucleotide conjugates and / or EV-associated target molecules comprising DNA and / or RNA in a sample (such as within and / or on an EV of a partitioned compartment). In some embodiments, the binding agent comprises an antibody, a knottin, an aptamer, an affimer, an avimer, a nanobody, a DARPin, a monobody, or an affinityAtty. Docket No. GH0250WO 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 binding agent comprises an antibody (such as an antibody specific for an EV-associated target molecule).

[0227] In some embodiments, the detecting comprises digital PCR, quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), sequencing, proximity ligation assay, and / or proximity extension assay. 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.

[0228] Some embodiments of the disclosed methods comprise detecting one or more extracellular vesicles, such as as a positive control. In some embodiments, detecting one or more extracellular vesicles comprises (a) contacting the sample with at least one binding agent-oligonucleotide conjugate (such as at least one antibody-oligonucleotide conjugate) comprising a binding agent (such as an antibody) specific for at least one extracellular vesicle-specific marker; and (b) before or after a step of partitioning as described herein, detecting a presence, absence, or level of the at least one extracellular vesicle-specific marker by amplifying and / or sequencing an oligonucleotide of the at least one binding agent-oligonucleotide conjugate (such as at least one antibody-oligonucleotide conjugate) comprising a binding agent (such as an antibody) specific for at least one extracellular vesicle-specific marker; thereby detecting the one or more extracellular vesicles in the sample. In some embodiments, detecting one or more extracellular vesicles comprises (a) contacting the sample with at least one binding agent-oligonucleotide conjugate (such as at least one antibody-oligonucleotide conjugate) comprising a binding agent (such as an antibody) specific for at least one extracellular vesicle-specific marker; and (b) before a step of partitioning as described herein, detecting a presence, absence, or level of the at least one extracellular vesicle-specific marker by amplifying and / or sequencing an oligonucleotide of the at least one binding agent-oligonucleotide conjugate (such as at least one antibody-oligonucleotide conjugate) comprising a binding agent (such as an antibody) specific for at least one extracellular vesicle-specific marker; thereby detecting the one or more extracellular vesicles in the sample. In some embodiments, detecting one or more extracellularAtty. Docket No. GH0250WO vesicles comprises (a) contacting the sample with at least one binding agent-oligonucleotide conjugate (such as at least one antibody-oligonucleotide conjugate) comprising an antibody specific for at least one extracellular vesicle-specific marker; and (b) after a step of partitioning as described herein, detecting a presence, absence, or level of the at least one extracellular vesicle-specific marker by amplifying and / or sequencing an oligonucleotide of the at least one binding agent-oligonucleotide conjugate (such as at least one antibody-oligonucleotide conjugate) comprising a binding agent (such as an antibody) specific for at least one extracellular vesicle-specific marker; thereby detecting the one or more extracellular vesicles in the sample.

[0229] In some embodiments, the detecting the presence, absence, or level of at least one extracellular vesicle-specific marker occurs after a step of partitioning and simultaneously with a step of detecting the presence, absence, or level of each of the plurality of EV-associated target molecules. In some embodiments, the at least one extracellular vesicle-specific marker comprises CD81, CD63, CD9, ALIX, CD40, Hsp60, TSG1, or phosphatidylserine. In some embodiments, the at least one extracellular vesicle-specific marker comprises CD81. In some embodiments, the at least one extracellular vesicle-specific marker comprises CD63. In some embodiments, the at least one extracellular vesicle-specific marker comprises CD9. In some embodiments, the at least one extracellular vesicle-specific marker comprises ALIX. In some embodiments, the at least one extracellular vesicle-specific marker comprises CD40. In some embodiments, the at least one extracellular vesicle-specific marker comprises Hsp60. In some embodiments, the at least one extracellular vesicle-specific marker comprises TSG1. In some embodiments, the at least one extracellular vesicle-specific marker comprises phosphatidylserine.

[0230] In some embodiments, the sample is contacted with the at least one binding agent-oligonucleotide conjugate (such as at least one antibody-oligonucleotide conjugate) comprising a binding agent (such as an antibody) specific for at least one extracellular vesicle-specific marker (i) before the sample is contacted with the plurality of binding agent-oligonucleotide conjugates (such as antibody-oligonucleotide conjugates), (ii) at the same time that the sample is contacted with the plurality of binding agent-oligonucleotide conjugates (such as antibody-oligonucleotide conjugates), or (iii) after the sample is contacted with the plurality of binding agent-oligonucleotide conjugates (such as antibody-oligonucleotide conjugates) and before the partitioning. In some embodiments, the sample is contacted with the at least one binding agent-oligonucleotide conjugate (such as at least one antibody-oligonucleotide conjugate) comprising a binding agent (such as an antibody) specific for at least one extracellular vesicle-specific markerAtty. Docket No. GH0250WObefore the sample is contacted with the plurality of antibody-oligonucleotide conjugates. In some embodiments, the sample is contacted with the at least one binding agent-oligonucleotide conjugate (such as at least one antibody-oligonucleotide conjugate) comprising a binding agent (such as an antibody) specific for at least one extracellular vesicle-specific marker at the same time that the sample is contacted with the plurality of binding agent-oligonucleotide conjugates (such as antibody-oligonucleotide conjugates). In some embodiments, the sample is contacted with the at least one binding agent-oligonucleotide conjugate (such as at least one antibody-oligonucleotide conjugate) comprising a binding agent (such as an antibody) specific for at least one extracellular vesicle-specific marker after the sample is contacted with the plurality of binding agent-oligonucleotide conjugates (such as antibody-oligonucleotide conjugates) and before the partitioning.

[0231] In some embodiments, at least a portion of the extracellular vesicles in the sample are isolated from other components of the sample using a different extracellular vesicle-specific marker than the at least one extracellular vesicle-specific marker used for detecting the one or more extracellular vesicles in the sample.1. Proximity Extension Assay; Proximity Ligation Assay

[0232] In particular embodiments, the detecting comprises a proximity ligation assay or proximity extension assay, wherein at least one of the plurality of binding agent-oligonucleotide conjugates (such as antibody-oligonucleotide conjugates) comprises a binding agent (such as an antibody) specific for at least one extracellular vesicle-specific marker. In some embodiments, the at least one extracellular vesicle-specific marker comprises one or more of CD81, CD63, CD9, ALIX, CD40, Hsp60, TSG1, or phosphatidylserine.

[0233] In some embodiments, the detecting comprises a proximity extension assay. In a proximity extension assay, first and second binding agents (such as first and second 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, such as one or more of CD81, CD63, CD9, ALIX, CD40, Hsp60, TSG1, or phosphatidylserine) 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., aAtty. Docket No. GH0250WO 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 the same EV-associated target molecule, 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 setp), thus indicating the presence of the EV-associated target molecule, e.g., on an EV within a partitioned compartment. Where the presence, absence, or level of a plurality of target EV-associated target molecules is being detected, the assay may be multiplexed.

[0234] In some embodiments, the detecting comprises a proximity ligation assay. In a proximity ligation assay, first and second binding agents (such as first and second 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 CD81, CD63, CD9, ALIX, CD40, Hsp60, TSG1, or phosphatidyl serine), 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 the same EV-associated target molecule, 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 of 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 the presence of the EV-associated target molecule, e.g., on an EV within a partitioned compartment. Where the presence, absence, or level of a plurality of target EV-associated target molecules is being detected, the assay may be multiplexed.Atty. Docket No. GH0250WO 2. Digital PCR

[0235] In some embodiments, the plurality of compartments is 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.

[0236] 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, 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 etal. 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-2020-0028.3. Sequencing

[0237] In some embodiments, the plurality of compartments is a plurality of droplets, and the detection step comprises sequencing (such as using next generation sequencing) oligonucleotides of the plurality of 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 from each partitioned compartment (e.g., each droplet) are obtained and analyzed in silico. After sequencing, analysis of reads, such as to identify oligonucleotides and / or detect genetic variantsAtty. Docket No. GH0250WOin 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 moleculeds 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.

[0238] 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, Maxim-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.

[0239] 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-timeAtty. Docket No. GH0250WO(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.

[0240] 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 read (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.

[0241] 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 silica 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 el al., FlOOOResearch, 6:100, 2017.) SMRTAtty. Docket No. GH0250WOsequencing 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.

[0242] 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.

[0243] 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 by 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.

[0244] 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 etal., 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.

[0245] 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 etal.. 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 moleculesAtty. Docket No. GH0250WO 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 nextgeneration 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 single-cell 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.4. Sequencing Methods with Partitioning

[0246] 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. For example, a sample from a subject can be partitioned into one or more subsamples, which can each be used to analyze one or more characteristics of the sample. In some embodiments, at least a first subsample is used in a method of analyzing a plurality of EV-associated target molecules as described elsewherein herein, and at least a second subsample is subjected to a sequencing method with partitioning as described in this section. Such partitioning can be used either for individual samples or pooled samples, in which nucleic acids from different samples are distinguished by sample indexes. Such 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. Such 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.

[0247] The number of such partitions depends on the characteristics of a population of nucleic acid molecules to be partitioned. These characteristics include the mean, median and mode of nucleic 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.Atty. Docket No. GH0250WO

[0248] 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.

[0249] 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.

[0250] 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.

[0251] 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 indexes 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 partitionAtty. Docket No. GH0250WOindexes and vice versa. Preferably the code designations of a set of sample and partition indexes are mutually exclusive with one another.

[0252] 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.

[0253] 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.

[0254] 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. Lane information, when determined, can also be used in grouping sequencing reads. Grouping of sequencing reads by molecule of origin permits distinction of genuine genetic or epigenetic variation from amplification and sequencing errors as further described below.Atty. Docket No. GH0250WO

[0255] 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).

[0256] 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-methylcytosine sequencing (SEM-seq) conversion, or direct methylation sequencing (DM-seq).

[0257] 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 different samples, partition the pooled samples, conversion of portions of the partitioned samples, amplification, enrichment and sequencing.Atty. Docket No. GH0250WO

[0258] 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.

[0259] 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.

[0260] 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 aliquot-specific 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 a 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 areAtty. Docket No. GH0250WO aliquot-specific), 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.

[0261] 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 sample demultiplexing is not required. Demultiplexing by sample of origin is based on the sample index or the sample-specific partition index.Atty. Docket No. GH0250WO G. Isolating

[0262] Some embodiments of the disclosed methods comprise isolating at least a portion of the extracellular vesicles in a sample from other components of the sample, thereby providing isolated extracellular vesicles. In some embodiments, the isolating comprises affinity purification, immunoprecipitation, size-exclusion chromatography, or centrifugation. In particular embodiments, the isolating comprises capturing extracellular vesicles in the sample using one or more extracellular vesicle-specific markers. In some embodiments, the isolating comprises capturing extracellular vesicles in the sample using one or more binding agents specific for one or more extracellular vesicle-specific markers. In some embodiments, the one or more binding agents is bound to a solid support. In some embodiments, the binding agent 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 extracellular vesicle-specific marker). In particular embodiments, the binding agent comprises an antibody (such as an antibody specific for an extracellular vesicle-specific marker).

[0263] In some embodiments, the one or more extracellular vesicle-specific marker comprises one or more of CD81, CD63, CD9, ALIX, CD40, Hsp60, TSG1, or phosphatidylserine. In some embodiments, the one or more extracellular vesicle-specific marker comprises CD81. In some embodiments, the one or more extracellular vesicle-specific marker comprises CD63. In some embodiments, the one or more extracellular vesicle-specific marker comprises CD9. In some embodiments, the one or more extracellular vesicle-specific marker comprises ALIX. In some embodiments, the one or more extracellular vesicle-specific marker comprises CD40. In some embodiments, the one or more extracellular vesicle-specific marker comprises Hsp60. In some embodiments, the one or more extracellular vesicle-specific marker comprises TSG1. In some embodiments, the one or more extracellular vesicle-specific marker comprises phosphatidyl serine. In some embodiments, the at least a portion of the extracellular vesicles in the sample are isolated from other components of the sample using a different extracellular vesicle-specific marker than at least one extracellular vesicle-specific marker used for detecting one or more extracellular vesicles in the sample.

[0264] In some embodiments, the step of isolating occurs prior to a step of contacting the sample with a plurality of binding agent-oligonucleotide conjugates (such as antibody-oligonucleotide conjugates) comprising at least (i) a first binding agent-oligonucleotide conjugate comprising aAtty. Docket No. GH0250WOfirst oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule. In some embodiments, the step of isolating occurs prior to a step of partitioning extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles. In some embodiments, the step of isolating occurs after a step of a step of contacting the sample with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule; and prior to a step of partitioning extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles. In particular embodiments, the binding agent comprises an antibody.

[0265] In some embodiments, EVs in the sample that are bound to the one or more binding agents (such as one or more 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.

[0266] The agents used to isolate EVs can be affinity agents, such as binding 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)). In some embodiments, EV-specific markers bound to an agent 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.

[0267] 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 captureing extracellular vesicles inAtty. Docket No. GH0250WOthe 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 agents (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.

[0268] 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., a binding agent (such as an antibody) labeled with a capture moiety, such as biotin or the other examples noted below. Binding agents (such as binding agents (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 agents (such as antibodies) specific for one or more EV-specific markers can have higher and lower capture yields for different EV-specific markers.

[0269] Capture may be performed using any suitable approach known in the art. EV capture can involve use of a binding agent (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.

[0270] 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 agent (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 antimouse secondary antibody. In some embodiments, the anti-mouse secondary antibody is a goat anti-mouse 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 asbiotin / streptavidin or hapten / antibody. In some embodiments, a capture moiety that is attached to a binding agent (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 anyAtty. Docket No. GH0250WOtype 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.

[0271] In some embodiments, the binding agents (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 moiety 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 agents (such as antibodies) are also eluted from the solid support by, e g., disrupting the biotinstreptavidin interaction.

[0272] 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 filtration 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.

[0273] 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 etAtty. Docket No. GH0250WO al., J TranslMed, 2021, 19:104, doi: 10.1186 / sl2967-021-02775-9; Gamez- Valero et al., Sci Rep, 2016, 6:33641, doi: 10.1038 / srep33641).H. Samples and Subjects

[0274] The disclosure relates to methods of analyzing a plurality of extracellular vesicle (EV)-associated target molecules in a sample. In some cases, the sample used in a method disclosed herein is obtained or has been obtained from a subject. In some embodiments, the sample is a biological sample obtained from a 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. Preferably, the sample is from a human. The subject may in some cases have or be suspected of having a cancer, tumor or neoplasm. In other cases, 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, e.g. from a tumor, cancer, or neoplasia (e g., following treatment such as chemotherapy, surgical resection, radiation, or a combination thereof). The subject may or may not be diagnosed as being susceptible to cancer or any cancer-associated genetic mutations / disorders. In some embodiments, the sample is a sample obtained from a tumor tissue biopsy. The cancer, tumor, or neoplasm may generally be of any type, for example a cancer tumor or neoplasm of the lung, colon, rectum (or colorectum), kidney, breast, prostate, or liver, or other type of cancer as described herein. 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 precancer, cancer, tumor, or neoplasia or suspected precancer, cancer, tumor, or neoplasia may be of the bladder, head and neck, lung, colon, rectum, kidney, breast, prostate, skin, or liver. In some embodiments, the precancer, cancer, tumor, or neoplasia or suspected precancer, cancer, tumor, or neoplasia is of the lung. In some embodiments, the precancer, cancer, tumor, or neoplasia or suspected precancer, cancer, tumor, or neoplasia is of the colon or rectum. In some embodiments, the precancer, cancer, tumor, or neoplasia or suspected precancer, cancer, tumor, or neoplasia is of the breast. In some embodiments, the precancer, cancer, tumor, or neoplasia or suspected precancer, cancer, tumor, or neoplasia is of the prostate. In any of the foregoingAtty. Docket No. GH0250WO embodiments, the subject may be a human subject. 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.

[0275] 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.

[0276] Biological samples can include body tissues, such as known or suspected solid tumors (such as carcinomas, adenocarcinomas, or sarcomas), 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, including gingival crevicular fluid, bone marrow, pleural effusions, cerebrospinal fluid, saliva, mucous, sputum, semen, sweat, and urine. In some embodiments, biological samples are body fluids, particularly blood and fractions thereof (e.g., plasma and / or serum) 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 extracellular vesicles, or enrich for one component relative to another.

[0277] 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.

[0278] In some embodiments, extracelluar vesicles comprising a plurality of EV-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.

[0279] 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.Atty. Docket No. GH0250WO

[0280] In a particular embodiment, the sample comprises extracellular vesicles.

[0281] 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 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 of being susceptible to cancer or any cancer-associated genetic mutations / disorders.

[0282] In some embodiments, the sample comprises plasma. The volume of plasma used to obtain the EVs can depend on the desired read depth for sequenced oligonucleotides or EV-associated nucleic acids. Exemplary volumes are 0.4-40 ml, 5-20 ml, 10-20 ml. For example, the volume can be 0.5 mL, 1 mb, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mb, 10 mL, 20 mb, 30 mL, or 40 mL. 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.

[0283] 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.

[0284] 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.

[0285] 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 sampled PBMCs 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.Atty. Docket No. GH0250WO

[0286] 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 mb, 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.

[0287] 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.

[0288] 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.I. Analysis

[0289] The present disclosure provides methods of analyzing a plurality of extracellular vesicle (EV)-associated target molecules in a sample. Methods of analyzing a plurality of EV-associated target molecules herein comprise (a) partitioning extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitionedAtty. Docket No. GH0250WO extracellular vesicles; and (b) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules using nucleic acid sequencing; optionally wherein the sample is contacted with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule, further optionally wherein the contacting occurs prior to step (a). In some embodiments, the binding agent comprises an antibody.

[0290] In some embodiments, the method comprises: (a) contacting the sample with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule, (iii) a third binding agent-oligonucleotide conjugate comprising a third oligonucleotide and a third binding agent specific for a third EV-associated target molecule, and (iv) a fourth binding agent-oligonucleotide conjugate comprising a fourth oligonucleotide and a fourth binding agent specific for a fourth EV-associated target molecule; (b) partitioning the extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles; and (c) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules by amplifying and / or sequencing oligonucleotides of the plurality of binding agent-oligonucleotide conjugates. In some embodiments, the binding agent comprises an antibody.

[0291] In some embodiments, the method comprises: (a) partitioning the extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles; and (b) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules, wherein the plurality of EV-associated target molecules comprise a plurality of EV-associated nucleic acids, wherein the detecting comprises nucleic acidAtty. Docket No. GH0250WOamplification and / or sequencing; optionally wherein the sample is contacted with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule, further optionally wherein the contacting occurs prior to step (a). In some embodiments, the binding agent comprises an antibody.

[0292] In some embodiments, the method comprises: (a) contacting the sample with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule; wherein the first oligonucleotide comprises a subsequence complementary to a first portion of the second oligonucleotide; (b) partitioning extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles; (c) before or after the partitioning, contacting the first and second oligonucleotides with a polymerase, wherein first and second oligonucleotides of binding agent-oligonucleotide conjugates that are bound to EV-associated target molecules of the same extracellular vesicle undergo extension to form extended oligonucleotides; and (d) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules using amplification of the extended oligonucleotides. In some embodiments, the binding agent comprises an antibody.

[0293] In some embodiments, the method comprises: (a) contacting the sample with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule; wherein the first oligonucleotide comprises a first subsequence complementary to a first portion of a connector oligonucleotide and the second oligonucleotide comprises a second subsequence complementary to a second portion of the connectorAtty. Docket No. GH0250WO oligonucleotide; (b) partitioning extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles; (c) before or after the partitioning, contacting the first and second oligonucleotides with a ligase in the presence of the connector oligonucleotides, wherein first and second oligonucleotides of binding agent-oligonucleotide conjugates that are bound to EV-associated target molecules of the same extracellular vesicle undergo ligation to form ligated oligonucleotides; and (d) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules using amplification of the ligated oligonucleotides. In some embodiments, the binding agent comprises an antibody.

[0294] The presenct methods can be used to detecting a presence, absence, or level of each of a plurality of EV-associated target molecules, such as using amplification and / or nucleic acid sequencing. The present methods can also be used to diagnose 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 decrease the level of each of the plurality of EV-associated target molecules detected in a subject’s blood if the treatment is successful as there will be fewer cancer cells to shed or release EVs. This correlation may be useful in selecting a therapy. In other examples, this may not occur.

[0295] 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.

[0296] 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, colon cancers, prostate cancers, thyroid cancers, bladder cancers, head and neck 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 changes in the presence, absence, and / or level of each of a plurality of EV-associated target molecules, such as genetic variations in EV-associated nucleic acids, including mutations, rare mutations, indels, copy number variations,Atty. Docket No. GH0250WOtransversions, 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.

[0297] In some embodiments, a method described herein comprises identifying the presence or level of EV-associated nucleic acids, such as DNA or RNA, produced by a tumor (or neoplastic cells, or cancer cells) or by precancer cells.

[0298] Genetic data can 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 useful 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 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.

[0299] 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 EV-associated target molecules in the subject, wherein the profile comprises a plurality of data resulting from genetic information (such as from sequencing EV-associated nucleic acids, such as RNA or DNA), such as copy number variation and rare mutation analyses, and / or the presence, absence, or level of EV-associated target molecules comprising proteins, such as EV-associated surface proteins. Tn 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. 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.

[0300] The present methods can thus be used to generate or profile, fingerprint or set of data that is a summation of nucleic acid and protein information derived from EVs in a heterogeneousAtty. Docket No. GH0250WO disease. This set of data may comprise protein information and / or genetic information, such as copy number variation, and / or rare mutation analyses, alone or in combination.

[0301] The present disclosure provides methods of analyzing a plurality of extracellular vesicle (EV)-associated target molecules in a sample. In some embodiments, detecting the presence, absence, or level of the plurality of EV-associated target molecules facilitates disease diagnosis or identification of appropriate treatments. In some embodiments, the presence of or a change in the levels of the plurality of EV-associated target molecules is indicative of the presence or absence of a disease or disorder in a subject, such as cancer or precancer, or other disorder that causes changes in the plurality of EV-associated target molecules relative to a healthy subject.

[0302] Information and data generated by the methods disclosed herein can also be used for characterizing a specific form of cancer. The methods disclosed herein 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.

[0303] Further, the methods of the disclosure may be used to characterize the heterogeneity of a condition in a subject. Such methods can include, e.g., generating an aggregate profile of a plurality of EV-associated target molecules derived from the subject, wherein the aggregate profile comprises a plurality of data resulting from various nucleic acid analyses, such as amplification and / or sequencing of oligonucleotides of binding agent-oligonucleotide conjugates (such as antibody-oligonucleotide conjugates) bound to the EVs, and / or amplification and / or sequencing of EV-associated nucleic acids.

[0304] An exemplary method for analyzing a plurality of EV-associated target molecules in a sample comprises the following steps, as illustrated in FIG. 1A:1. Optionally isolating at least a portion of the extracellular vesicles in the sample (such as a blood sample) from other components of the sample, thereby providing isolated extracellular vesicles.2. Optionally contacting the sample with a plurality of binding agent-oligonucleotide conjugates (such as a plurality of antibody-oligonucleotide conjugates) comprising at least (i) aAtty. Docket No. GH0250WO first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule.3. Partitioning extracellular vesicles in the sample into a plurality of compartments (such as a plurality of droplets), wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles.4. For at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules using nucleic acid sequencing, such as NGS sequencing.

[0305] Another exemplary method for analyzing a plurality of EV-associated target molecules in a sample comprises the following steps, as illustrated in FIG. IB:1. Optionally isolating at least a portion of the extracellular vesicles in the sample (such as a blood sample) from other components of the sample, thereby providing isolated extracellular vesicles.2. Contacting the sample (such as a blood sample) with a plurality of binding agent-oligonucleotide conjugates (such as a plurality of antibody-oligonucleotide conjugates) comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule, (iii) a third binding agent-oligonucleotide conjugate comprising a third oligonucleotide and a third binding agent specific for a third EV-associated target molecule, and (iv) a fourth binding agent-oligonucleotide conjugate comprising a fourth oligonucleotide and a fourth binding agent specific for a fourth EV-associated target molecule.3. Partitioning the extracellular vesicles in the sample into a plurality of compartments (such as a plurality of droplets), wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles.4. For at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules by amplifying and / orAtty. Docket No. GH0250WOsequencing (such as using digital PCR or NGS sequencing) oligonucleotides of the plurality of binding agent-oligonucleotide conjugates.

[0306] Another exemplary method for analyzing a plurality of EV-associated target molecules in a sample comprises the following steps, as illustrated in FIG. 1C:1. Optionally isolating at least a portion of the extracellular vesicles in the sample (such as a blood sample) from other components of the sample, thereby providing isolated extracellular vesicles.2. Optionally contacting the sample with a plurality of binding agent-oligonucleotide conjugates (such as a plurality of antibody-oligonucleotide conjugates) comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule.3. Partitioning the extracellular vesicles in the sample into a plurality of compartments (such as a plurality of droplets), wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles.4. For at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules, wherein the plurality of EV-associated target molecules comprise a plurality of EV-associated nucleic acids, wherein the detecting comprises nucleic acid amplification and / or sequencing (such as digital PCR or NGS sequencing).

[0307] Another exemplary method for analyzing a plurality of EV-associated target molecules in a sample comprises the following steps, as illustrated in FIG. ID:1. Optionally isolating at least a portion of the extracellular vesicles in the sample (such as a blood sample) from other components of the sample, thereby providing isolated extracellular vesicles.2. Contacting the sample with a plurality of binding agent-oligonucleotide conjugates (such as a plurality of antibody-oligonucleotide conjugates) comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a secondAtty. Docket No. GH0250WOEV-associated target molecule; wherein the first oligonucleotide comprises a subsequence complementary to a first portion of the second oligonucleotide.3. Partitioning extracellular vesicles in the sample into a plurality of compartments (such as a plurality of droplets), wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles.4. Before the partitioning, contacting the first and second oligonucleotides with a polymerase, wherein first and second oligonucleotides of binding agent-oligonucleotide conjugates that are bound to EV-associated target molecules of the same extracellular vesicle undergo extension to form extended oligonucleotides.5. For at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules using amplification of the extended oligonucleotides.

[0308] Another exemplary method for analyzing a plurality of EV-associated target molecules in a sample comprises the following steps, as illustrated in FIG. IE:1. Optionally isolating at least a portion of the extracellular vesicles in the sample (such as a blood sample) from other components of the sample, thereby providing isolated extracellular vesicles.2. Contacting the sample with a plurality of binding agent-oligonucleotide conjugates (such as a plurality of antibody-oligonucleotide conjugates) comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule; wherein the first oligonucleotide comprises a subsequence complementary to a first portion of the second oligonucleotide.3. Partitioning extracellular vesicles in the sample into a plurality of compartments (such as a plurality of droplets), wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles.4. After the partitioning, contacting the first and second oligonucleotides with a polymerase, wherein first and second oligonucleotides of binding agent-oligonucleotide conjugates that are bound to EV-associated target molecules of the same extracellular vesicle undergo extension toAtty. Docket No. GH0250WO form extended oligonucleotides.5. For at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules using amplification of the extended oligonucleotides.

[0309] Another exemplary method for analyzing a plurality of EV-associated target molecules in a sample comprises the following steps, as illustrated in FIG. IF:1. Optionally isolating at least a portion of the extracellular vesicles in the sample (such as a blood sample) from other components of the sample, thereby providing isolated extracellular vesicles.2. Contacting the sample with a plurality of binding agent-oligonucleotide conjugates (such as a plurality of antibody-oligonucleotide conjugates) comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule; wherein the first oligonucleotide comprises a first subsequence complementary to a first portion of a connector oligonucleotide and the second oligonucleotide comprises a second subsequence complementary to a second portion of the connector oligonucleotide.3. Partitioning extracellular vesicles in the sample into a plurality of compartments (such as a plurality of droplets), wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles.4. Before the partitioning, contacting the first and second oligonucleotides with a ligase in the presence of the connector oligonucleotides, wherein first and second oligonucleotides of binding agent-oligonucleotide conjugates that are bound to EV-associated target molecules of the same extracellular vesicle undergo ligation to form ligated oligonucleotides.5. For at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules using amplification of the ligated oligonucleotides.

[0310] Another exemplary method for analyzing a plurality of EV-associated target molecules in a sample comprises the following steps, as illustrated in FIG. 1G:1. Optionally isolating at least a portion of the extracellular vesicles in the sample (such as aAtty. Docket No. GH0250WO blood sample) from other components of the sample, thereby providing isolated extracellular vesicles.2. Contacting the sample with a plurality of binding agent-oligonucleotide conjugates (such as a plurality of antibody-oligonucleotide conjugates) comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule; wherein the first oligonucleotide comprises a first subsequence complementary to a first portion of a connector oligonucleotide and the second oligonucleotide comprises a second subsequence complementary to a second portion of the connector oligonucleotide.3. Partitioning extracellular vesicles in the sample into a plurality of compartments (such as a plurality of droplets), wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles.4. After the partitioning, contacting the first and second oligonucleotides with a ligase in the presence of the connector oligonucleotides, wherein first and second oligonucleotides of binding agent-oligonucleotide conjugates that are bound to EV-associated target molecules of the same extracellular vesicle undergo ligation to form ligated oligonucleotides.5. For at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules using amplification of the ligated oligonucleotides.J. Computer Systems

[0311] Methods of the present disclosure can be implemented using, or with the aid of, computer systems. FIG. 2 shows a computer system 201 that is programmed or otherwise configured to implement the methods of the present disclosure. The computer system 201 can regulate various aspects sample preparation, sequencing, and / or analysis. In some examples, the computer system 201 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.

[0312] The computer system 201 includes a central processing unit (CPU, also "processor" and "computer processor" herein) 205, which can be a single core or multi core processor, or aAtty. Docket No. GH0250WOplurality of processors for parallel processing. The computer system 201 also includes memory or memory location 210 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 215 (e.g., hard disk), communication interface 220 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 225, such as cache, other memory, data storage, and / or electronic display adapters. The memory 210, storage unit 215, interface 220, and peripheral devices 225 are in communication with the CPU 205 through a communication network or bus (solid lines), such as a motherboard. The storage unit 215 can be a data storage unit (or data repository) for storing data. The computer system 201 can be operatively coupled to a computer network 230 with the aid of the communication interface 220. The computer network 230 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 230 in some cases is a telecommunication and / or data network. The computer network 230 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The computer network 230, in some cases with the aid of the computer system 201, can implement a peer-to-peer network, which may enable devices coupled to the computer system 201 to behave as a client or a server.

[0313] The CPU 205 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 210. Examples of operations performed by the CPU 205 can include fetch, decode, execute, and writeback.

[0314] The storage unit 215 can store files, such as drivers, libraries, and saved programs. The storage unit 215 can store programs generated by users and recorded sessions, as well as output(s) associated with the programs. The storage unit 215 can store user data, e.g., user preferences and user programs. The computer system 201 in some cases can include one or more additional data storage units that are external to the computer system 201 , such as located on a remote server that is in communication with the computer system 201 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).

[0315] The computer system 201 can communicate with one or more remote computer systems through the network 230. For embodiment, the computer system 201 can communicate with a remote computer system of a user (e.g., operator). Examples of remote computer systems includeAtty. Docket No. GH0250WOpersonal 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 201 via the network 230.

[0316] 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 201, such as, for example, on the memory 210 or electronic storage unit 215. 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 205. In some cases, the code can be retrieved from the storage unit 215 and stored on the memory 210 for ready access by the processor 205. In some situations, the electronic storage unit 215 can be precluded, and machine-executable instructions are stored on memory 210.

[0317] 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: (a) partitioning extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles; and (b) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules using nucleic acid sequencing; optionally wherein the sample is contacted with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule, further optionally wherein the contacting occurs prior to step (a).

[0318] In another example, the method may comprise: (a) contacting the sample with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule, (iii) a third binding agent-oligonucleotide conjugate comprising aAtty. Docket No. GH0250WO third oligonucleotide and a third binding agent specific for a third EV-associated target molecule, and (iv) a fourth binding agent-oligonucleotide conjugate comprising a fourth oligonucleotide and a fourth binding agent specific for a fourth EV-associated target molecule; (b) partitioning the extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles; and (c) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules by amplifying and / or sequencing oligonucleotides of the plurality of binding agent-oligonucleotide conjugates.

[0319] In another example, the method may comprise: (a) partitioning the extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles; and (b) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules, wherein the plurality of EV-associated target molecules comprise a plurality of EV-associated nucleic acids, wherein the detecting comprises nucleic acid amplification and / or sequencing; optionally wherein the sample is contacted with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule, further optionally wherein the contacting occurs prior to step (a).

[0320] In another example, the method may comprise (a) contacting the sample with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule; wherein the first oligonucleotide comprises a subsequence complementary to a first portion of the second oligonucleotide; (b) partitioning extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles; (c) before or after the partitioning,Atty. Docket No. GH0250WO contacting the first and second oligonucleotides with a polymerase, wherein first and second oligonucleotides of binding agent-oligonucleotide conjugates that are bound to EV-associated target molecules of the same extracellular vesicle undergo extension to form extended oligonucleotides; and (d) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules using amplification of the extended oligonucleotides.

[0321] In another example, the method may comprise (a) contacting the sample with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule; wherein the first oligonucleotide comprises a first subsequence complementary to a first portion of a connector oligonucleotide and the second oligonucleotide comprises a second subsequence complementary to a second portion of the connector oligonucleotide; (b) partitioning extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles; (c) before or after the partitioning, contacting the first and second oligonucleotides with a ligase in the presence of the connector oligonucleotides, wherein first and second oligonucleotides of binding agent-oligonucleotide conjugates that are bound to EV-associated target molecules of the same extracellular vesicle undergo ligation to form ligated oligonucleotides; and (d) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules using amplification of the ligated oligonucleotides.

[0322] 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.

[0323] Aspects of the systems and methods provided herein, such as the computer system 201, 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 machineAtty. Docket No. GH0250WOreadable 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 for the software programming.

[0324] All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical, and electromagnetic waves, such as those used across physical interfaces between local devices, through wired and optical landline networks, and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links, or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible "storage" media, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution.

[0325] Hence, a machine-readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards, paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any otherAtty. Docket No. GH0250WO medium from which a computer may read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0326] The computer system 201 can include or be in communication with an electronic display 235 that comprises a user interface (UI) 240 for providing, for example, one or more results of sample analysis. Examples of UIs include, without limitation, a graphical user interface (GUI) and web-based user interface.

[0327] Additional details relating to computer systems and networks, databases, and computer program products are also provided in, for example, Peterson, Computer Networks: A Systems Approach, Morgan Kaufmann’ 5th Ed. (2011), Kurose, Computer Networking: A Top-Down Approach, Pearson, 7thEd. (2016), Elmasri, Fundamentals of Database Systems, Addison Wesley 6th Ed. (2010), Coronel, Database Systems: Design, Implementation, & Management, Cengage Learning, 11thEd. (2014), Tucker, Programming Languages, McGraw-Hill Science / Engineering / Math’ 2nd Ed. (2006), and Rhoton, Cloud Computing Architected: Solution Design Handbook, Recursive Press (2011), each of which is hereby incorporated by reference in its entirety.K. Applications

[0328] 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.

[0329] 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-associated 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.

[0330] Hence, in some embodiments, methods described herein comprise identifying or predicting the presence or absence of proteins or nucleic acids produced by a tumor (orAtty. Docket No. GH0250WO 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

[0331] 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 determining 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 rare mutations associated with EVs in a subject's blood if the treatment is successful as more cancers may die and shed or release EVs. In other examples, this may not occur. In another example, certain treatment options may be correlated with genetic profiles of cancers over time. This correlation may be useful in selecting a therapy. In some embodiments, successful treatment options may result in increases or decreases in the levels of EV-associated target molecules, such as proteins or nucleic acids, such as changes in copy number variation, rare mutations, and / or cancer-related epigenetic signatures (such as hypermethylated regions or hypomethylated regions) detected in, e.g., EVs in a sample from a subject, such as detected in EVs from a subject's blood (e.g., a whole blood sample, a plasma 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, or, e.g., if a successful treatment results in an increase or decrease in the quantity of a specific EV-associated target molecule and an unsuccessful treatment results in no change. In another example, certain treatment options may be correlated with genetic and / or associated protein profiles of EVs from cancers over time.

[0332] 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.Atty. Docket No. GH0250WO

[0333] In some embodiments, the present methods are used for screening for a cancer, such as a metastasis, or in a method for screening cancer, such as in a method of detecting the presence or absence of a metastasis. For example, the sample can be a sample from a subject who has or has not been previously diagnosed with cancer. 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. 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.

[0334] 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 / Di etary_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 mL 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 half-sibling), 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.

[0335] 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 the 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 includeAtty. Docket No. GH0250WO biliary tract cancer, bladder cancer, transitional cell carcinoma, urothelial carcinoma, brain cancer, gliomas, astrocytomas, breast cancer, 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, lymphocytic cancer, 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, lymphoma, 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, myeloma, 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.

[0336] In some embodiments, the cancer is a type of cancer that is not a hematological cancer, e.g., a solid tumor cancer such as a carcinoma, adenocarcinoma, or 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, such as 5mC and 5mC profiles. Hence, the present methods can in some cases be used in combination with methods used to detect other genetic / epigenetic variations, e.g. in a method of detecting or characterizing a cancer or other methods described herein. In some embodiments, a methodAtty. Docket No. GH0250WOdescribed herein comprises identifying the presence of target regions and / or DNA produced by a tumor (or neoplastic cells, or cancer cells) or by precancer cells. In some embodiments, a method described herein comprises determining the level of target regions and / or identifying the presence of DNA produced by a tumor (or neoplastic cells, or cancer cells) or by precancer cells. In some embodiments, determining the level of target regions comprises determining either an increased level or decreased level of target regions, wherein the increased or decreased level of target regions is determined by comparing the level of target regions with a threshold level / value.

[0337] Genetic and / or epigenetic data can also be used for characterizing a specific form of cancer. Cancers are often heterogeneous in both composition and staging. Genetic and / or epigenetic 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.

[0338] 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 and / or transcriptomic profile of nucleic acids associated with EVs from the subject, wherein the genetic and / or transcriptomic profile comprises a plurality of data resulting from copy number variation and rare mutation analyses, and / or levels of EV-associated RNAs. In some embodiments, an abnormal condition is cancer, e.g., as described herein. 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, such as where one or more foci (such as one or more tumor foci) are the result of metastases that have spread from a primary site of a cancer. The tissue(s) of origin can be useful for identifying organs affected by the cancer, including the primary cancer and / or metastatic tumors.

[0339] In some embodiments, sequencing oligonucleotides and / or EV-associated nucleic acids comprises generating a plurality of sequencing reads. Sequence information obtained in the present methods may comprise sequence reads of the oligonucleotides and / or nucleic acids generated by a nucleic acid sequencer. In some embodiments, the nucleic acid sequencerAtty. Docket No. GH0250WO performs pyrosequencing, single-molecule sequencing, nanopore sequencing, semiconductor sequencing, sequencing-by-synthesis, 5-letter sequencing, 6-letter sequencing, sequencing-by-ligation or sequencing-by-hybridization on the nucleic acids to generate sequencing reads. In some embodiments, the method further comprises mapping the plurality of sequence reads to one or more reference sequences to generate mapped sequence reads. In some embodiments, the method further comprises grouping the sequence reads into families of sequence reads, each family comprising sequence reads generated from a nucleic acid in the sample. In some embodiments, the methods comprise determining the likelihood that the subject from which the sample was obtained has cancer or precancer, or has a metastasis, that is related to changes in proportions of types of immune cells. In some embodiments, the methods comprises processing the mapped sequence reads to determine the likelihood that the subject has cancer or precancer.

[0340] The present methods can be used to generate or profile, fingerprint or set of data that is a summation of genetic and / or transcriptomic information derived from EVs in a heterogeneous disease. This set of data may comprise copy number variation, mutation analyses, and EV-associated RNA levels, alone or in combination.

[0341] 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 EVs and may co-circulate with maternal molecules.

[0342] 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,Atty. Docket No. GH0250WO trimethylaminuria, Turner syndrome, velocardiofacial syndrome, WAGR syndrome, Wilson disease, or the like.

[0343] 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. In some embodiments, a method described herein comprises detecting a presence or absence of EVs and EV-associated target molecules originating or derived from a tumor cell at a preselected timepoint following a previous cancer treatment of a subject previously diagnosed with cancer using a set of sequence information obtained as described herein. The method may further comprise determining a cancer recurrence score that is indicative of the presence or absence of the EVs and EV-associated target molecules originating or derived from the tumor cell for the subject.

[0344] 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 below 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.

[0345] 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.

[0346] 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 / orAtty. Docket No. GH0250WOclassifying a test subject as being a candidate for a subsequent cancer treatment

[0347] 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 or comprises a method of detecting the presence of absence of a metastasis in a subject. In some embodiments, a method provided herein is or comprises a method of classifying a subject as being a candidate for a subsequent cancer treatment.

[0348] Any of such methods may comprise collecting a sample (such as EVs originating or derived from a tumor cell) 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, such as a sample comprising EVs, may be a tissue sample or a blood sample (e.g., a whole blood sample, a buffy coat sample, a leukapheresis sample, or a PBMC sample).

[0349] In any of such methods, the previous cancer treatment may comprise surgery, administration of a therapeutic composition, and / or chemotherapy.

[0350] Any of such methods may comprise detecting a presence or absence or level of EV-associated target molecules 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 EV-associated target molecules, such as EV-associated proteins or nucleic acids originating or derived from a tumor cell may be performed according to any of the embodiments thereof described elsewhere herein.

[0351] 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 the EV-associated target molecules, such as RNAs or genomic regions of interest and target regions, 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.

[0352] 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 theAtty. Docket No. GH0250WOtissue-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.

[0353] 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 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. In some embodiments, the subsequent cancer treatment comprises chemotherapy or administration of a therapeutic composition.

[0354] 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.

[0355] In some embodiments, determining the cancer recurrence score may comprise determining at least a first subscore indicative of the amount of the levels of particular EV-associated target molecules, such as proteins, or nucleic acids comprising SNVs, insertions / deletions, CNVs and / or fusions.

[0356] In some embodiments, a number of mutations in the EV-associated nucleic acids 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.

[0357] In some embodiments, any of such methods may comprise determining a fraction of EV-associated target molecules that indicate one or more features indicative of origination from a tumor cell. This may be done for EV-associated target molecules that are proteins (such as EV surface proteins), or that are EV-associated nucleic acids comprising alterations consistent with cancer, such as SNVs, indels, CNVs, and / or fusions.

[0358] Determination of a cancer recurrence score may be based at least in part on the fraction of EV-associated tumor DNA, wherein a fraction of EV-associated tumor DNA greater than a threshold in the range of 10'11to 1 or IO'10to 1 is sufficient for the cancer recurrence score to be classified as positive for cancer recurrence. In some embodiments, a fraction of EV-associatedAtty. Docket No. GH0250WO tumor DNA greater than or equal to a threshold in the range of 1010to IO9, 109to 10s, 10sto IO7, 107to IO6, 106to 10s, 10sto IO4, 104to IO4, IO3to IO2, or 102to 101is sufficient for the cancer recurrence score to be classified as positive for cancer recurrence. In some embodiments, the fraction of EV-associated tumor DNA greater than a threshold of at least 10'7is sufficient for the cancer recurrence score to be classified as positive for cancer recurrence. A determination that a fraction of EV-associated tumor DNA is greater than a threshold, such as a threshold corresponding to any of the foregoing embodiments, may be made based on a cumulative probability. For example, the sample was considered positive if the cumulative probability that the tumor fraction was greater than a threshold in any of the foregoing ranges exceeds a probability threshold of at least 0.5, 0.75, 0.9, 0.95, 0.98, 0.99, 0.995, or 0.999. In some embodiments, the probability threshold is at least 0.95, such as 0.99.

[0359] In some embodiments, determining the cancer recurrence score comprises determining a subscore indicative of the amount of EV-associated proteins and a second subscore indicative of EV-associated nucleic acids comprising SNVs, insertions / deletions, CNVs and / or fusions and combining the subscores to provide the cancer recurrence score. Where the subscores are combined, they may be combined by applying a threshold to each subscore independently, or training a machine learning classifier to determine status based on a plurality of positive and negative training samples.

[0360] In some embodiments, a value for the combined score in the range of -4 to 2 or -3 to 1 is sufficient for the cancer recurrence score to be classified as positive for cancer recurrence.

[0361] 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.

[0362] 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

[0363] 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),Atty. Docket No. GH0250WO 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, monitoring comprises analysis of at least two samples collected from a subject at at least two different time points as described herein.

[0364] The methods according to the present disclosure can be useful in predicting a subject’s response to a particular treatment option, such as over a period of time. As described elsewhere herein, successful treatment options may increase the amount of cancer associated EVs in a subject's blood, such as if the treatment is successful as more cancers may die and shed or release EVs. In some examples, certain treatment options may be correlated with cancer EV-associated target molecule profiles (such as EV transcriptomic and / or genetic profiles) over time. This correlation may be useful in selecting a therapy.

[0365] 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 1-5, 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 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.

[0366] 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.Atty. Docket No. GH0250WO

[0367] In some embodiments of the disclosed methods, one or more samples (such as one or more tissue, 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.

[0368] In other embodiments of the disclosed methods, one or more samples (such as one or more tissue samples or blood samples, e.g., or 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, 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).4. Therapies and Related Administration

[0369] In certain embodiments, the methods disclosed herein relate to identifying and administering therapies, such as customized therapies, to patients. In some embodiments, determination of the levels of particular immune cell types, including rare immune cell types, facilitates selection of appropriate treatment. 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 chemotherapyAtty. Docket No. GH0250WO 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 and Fludarabine), plant alkaloids and natural products (for example, but not limited to, Vincristine, Paclitaxel and Topotecan), antitumor 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 some embodiments, the therapies are PARP inhibitors, such as Olaparib (LYNPARZA®), Rucaparib (RUB RAC A®), Niraparib (ZEJULA®), and Talazoparib (TALZENNA®). These may be used for treating mutations inBRCAl, BRCA2, ATM, BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCL, PALB2, RAD51B,RAD51 C, RAD51D and RAD54L alterations, and / or for genes associated Homologous Recombination Repair (HRR). 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.

[0370] 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 can 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.

[0371] 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.Atty. Docket No. GH0250WO

[0372] 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. Checkpoint 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, CD 137, KIR, CCR5, CD27, CD40, or CD47. Other exemplary agents include proinflammatory cytokines, such as IL-ip, IL-6, and TNF-or 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).

[0373] 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 (aka B7.1) 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 antitumor 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 3Atty. Docket No. GH0250WO (LAG3), killer cell immunoglobulin like receptor (KIR), T cell membrane protein 3 (TIM3), galectin 9 (GAL9), or adenosine A2a receptor (A2aR).

[0374] 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, the 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®).

[0375] In certain embodiments, the immunotherapy or immunotherapeutic agent is an antagonist (e.g. antibody) against CD80, CD86, LAG3, KIR, TIN , 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.

[0376] In some embodiments, the therapies target mutated forms of the EGFR protein. Such therapies can include osimertinib (TAGRISSO®), erlotinib (TARCEVA®), and gefinitib (IRESSA®).

[0377] Therapies can include one or more of treatments for target therapies, including abemaciclib (VERZENIO®), abiraterone acetate (ZYTIGA®), acalabrutinib (CALQUENCE®),Atty. Docket No. GH0250WOadagrasib (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®), binimetinib (MEKTOVI®), blinatumomab (BLINCYTO®), bortezomib (VELCADE®), bosutinib (BOSULIF®), brentuximab vedotin (ADCETRIS®), brexucabtagene autoleucel (TEC ARTUS®), 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 (TYKERB®), larotrectinib sulfate (VITRAKVI®), lenvatinib mesylateAtty. Docket No. GH0250WO(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 D APT®), mirvetuximab soravtansine-gynx (ELAHERE®), mobocertinib succinate (EXKIVITY®), mogamulizumab-kpkc (POTELIGEO®), mosunetuzumab-axgb (LUNSUMIO®), moxetumomab pasudotox-tdfk (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 (RUB RAC A®), 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 (ZMJUDO®), tretinoin (VESANOID®), tucatinib (TUKYSA®), vandetanib (CAPRELSA®), vemurafenib (ZELBORAF®), venetoclax (VENCLEXTA®), vismodegibAtty. Docket No. GH0250WO(ERIVEDGE®), vorinostat (ZOLINZA®), zanubrutinib (BRUKINSA®), and / or ziv-aflibercept (ZALTRAP®).

[0378] Table 7 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 7 including a mutation in one or more target genes listed in Table 7 for that cancer type, and the therapy administered to the subject comprises the drug listed in Table 7 for that cancer type and mutation.

[0379] Table 7. Exemplary drugsAty. Docket No. GH0250WOAty. Docket No. GH0250WOAty. Docket No. GH0250WOAty. Docket No. GH0250WOAty. Docket No. GH0250WOAty. Docket No. GH0250WOAty. Docket No. GH0250WOAtty. Docket No. GH0250WO

[0380] 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 7; and (ii) administering the corresponding one or more drugs listed in Table 7. In some embodiments, these therapies may be used alone or in combination with other therapies to treat a disease.Atty. Docket No. GH0250WO

[0381] 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. In 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.

[0382] Agonists that target these co-stimulatory checkpoint molecules can be used to enhance antigen-specific T cell responses against certain cancers. Accordingly, in certain embodim...

Claims

Atty. Docket No. GH0250WOWhat is claimed is:

1. A method of analyzing a plurality of extracellular vesicle (EV)-associated target molecules in a sample, the method comprising:a) partitioning extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles; andb) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules using nucleic acid sequencing;optionally wherein the sample is contacted with a plurality of binding agent- oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule, further optionally wherein the contacting occurs prior to step (a).

2. A method of analyzing a plurality of extracellular vesicle (EV)-associated target molecules in a sample, the method comprising:a) contacting the sample with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule, (iii) a third binding agent-oligonucleotide conjugate comprising a third oligonucleotide and a third binding agent specific for a third EV-associated target molecule, and (iv) a fourth binding agent-oligonucleotide conjugate comprising a fourth oligonucleotide and a fourth binding agent specific for a fourth EV-associated target molecule;b) partitioning the extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is presentAtty. Docket No. GH0250WOin at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles; andc) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules by amplifying and / or sequencing oligonucleotides of the plurality of binding agent-oligonucleotide conjugates.

3. A method of analyzing a plurality of extracellular vesicle (EV)-associated target molecules in a sample, the method comprising:a) partitioning the extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles; andb) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules, wherein the plurality of EV-associated target molecules comprise a plurality of EV-associated nucleic acids, wherein the detecting comprises nucleic acid amplification and / or sequencing; optionally wherein the sample is contacted with a plurality of binding agent- oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV-associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule, further optionally wherein the contacting occurs prior to step (a).

4. A method of analyzing a plurality of extracellular vesicle (EV)-associated target molecules in a sample, the method comprising:a) contacting the sample with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV- associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a secondAtty. Docket No. GH0250WO EV-associated target molecule; wherein the first oligonucleotide comprises a subsequence complementary to a first portion of the second oligonucleotide;b) partitioning extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles;c) before or after the partitioning, contacting the first and second oligonucleotides with a polymerase, wherein first and second oligonucleotides of binding agent- oligonucleotide conjugates that are bound to EV-associated target molecules of the same extracellular vesicle undergo extension to form extended oligonucleotides; and d) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules using amplification of the extended oligonucleotides.

5. A method of analyzing a plurality of extracellular vesicle (EV)-associated target molecules in a sample, the method comprising:a) contacting the sample with a plurality of binding agent-oligonucleotide conjugates comprising at least (i) a first binding agent-oligonucleotide conjugate comprising a first oligonucleotide and a first binding agent specific for a first EV- associated target molecule, and (ii) a second binding agent-oligonucleotide conjugate comprising a second oligonucleotide and a second binding agent specific for a second EV-associated target molecule; wherein the first oligonucleotide comprises a first subsequence complementary to a first portion of a connector oligonucleotide and the second oligonucleotide comprises a second subsequence complementary to a second portion of the connector oligonucleotide;b) partitioning extracellular vesicles in the sample into a plurality of compartments, wherein after the partitioning, only a single extracellular vesicle is present in at least a portion of the plurality of compartments, thereby providing partitioned extracellular vesicles;c) before or after the partitioning, contacting the first and second oligonucleotides with a ligase in the presence of the connector oligonucleotides, wherein first and second oligonucleotides of binding agent-oligonucleotide conjugates that are bound to EV-Atty. Docket No. GH0250WOassociated target molecules of the same extracellular vesicle undergo ligation to form ligated oligonucleotides; andd) for at least a portion of the partitioned extracellular vesicles, detecting a presence, absence, or level of each of the plurality of EV-associated target molecules using amplification of the ligated oligonucleotides.

6. The method of any one of the preceding claims, wherein the plurality of binding agent- oligonucleotide conjugates further comprises at least a third binding agent- oligonucleotide conjugate comprising a third binding agent specific for a third EV- associated target molecule.

7. The method of the immediately preceding claim, wherein the plurality of binding agent- oligonucleotide conjugates further comprises at least a fourth binding agent- oligonucleotide conjugate comprising a fourth binding agent specific for a fourth EV- associated target molecule.

8. The method of the immediately preceding claim, wherein the plurality of binding agent- oligonucleotide conjugates further comprises at least a fifth binding agent- oligonucleotide conjugate comprising a fifth binding agent specific for a fifth EV- associated target molecule.

9. The method of any one of the preceding claims, wherein the plurality of binding agent- oligonucleotide conjugates comprises 2-30, such as 2-25, 2-20, 2-15, 2-10, 2-5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more than 30 binding agent-oligonucleotide conjugates.

10. The method of any one of the preceding claims, wherein the binding agent comprises an antibody, a knottin, an aptamer, an affimer, an avimer, a nanobody, a DARPin, a monobody, or an affinity clamp.

11. The method of any one of the preceding claims, the binding agent comprises an antibody.Atty. Docket No. GH0250WO 12. The method of any one of the preceding claims, wherein the plurality of EV-associated target molecules comprises 2-30, such as 2-25, 2-20, 2-15, 2-10, 2-5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more than 30 EV-associated target molecules.

13. The method of any one of the preceding claims, wherein the plurality of EV-associated target molecules comprises one or more proteins.

14. The method of the immediately preceding claim, wherein the one or more proteins comprise one or more extracellular vesicle surface proteins.

15. The method of any one of the preceding claims, wherein the plurality of EV-associated target molecules comprises one or more EV-associated target nucleic acids.

16. The method of the immediately preceding claim, wherein the plurality of EV-associated target nucleic acids comprises one or more DNAs.

17. The method of claim 15 or 16, wherein the plurality of EV-associated target nucleic acids comprises one or more RNAs.

18. The method of the immediately preceding claim, wherein the one or more RNAs comprise one or more messenger RNAs (mRNA).

19. The method of any one of claims 17-18, wherein the one or more RNAs comprise one or more ribosomal RNAs (rRNAs).

20. The method of any one of claims 17-19, wherein the one or more RNAs comprise one or more transfer RNAs (tRNAs).

21. The method of any one of claims 17-20, wherein the one or more RNAs comprise one or more microRNAs (miRNAs).Atty. Docket No. GH0250WOl. The method of any one of claims 17-21, wherein the one or more RNAs comprise one or more small nuclear RNAs (snRNAs).

23. The method of any one of claims 17-22, wherein the one or more RNAs comprise one or more small interfering RNAs (siRNAs).

24. The method of any one of claims 17-23, wherein the one or more RNAs comprise one or more long non-coding RNAs (IncRNAs).

25. The method of any one of claims 17-24, wherein the method comprises amplifying the plurality of EV-associated target nucleic acids.

26. The method of any one of claims 17-25, wherein one or more barcodes are added to the plurality of EV-associated target nucleic acids or amplification products thereof.

27. The method of any one of the preceding claims wherein the one or more oligonucleotides comprises at least one tag.

28. The method of the immediately preceding claim, wherein the at least one tag comprises a molecular barcode.

29. The method of any one of the preceding claims, wherein the method comprises amplifying the one or more oligonucleotides.

30. The method of claims 25-29, wherein the amplifying comprises PCR, reverse transcription PCR (RT-PCR), rolling circle amplification, or linear amplification.

31. The method of any one of claims 25-30, wherein the amplifying occurs after the partitioning and prior to the detecting.

32. The method of any one of the preceding claims, wherein the plurality of compartments is a plurality of droplets.Atty. Docket No. GH0250WO33. The method of any one of the preceding claims, wherein the detecting comprises digital PCR, quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), sequencing, proximity ligation assay, and / or proximity extension assay.

34. The method of any one of the preceding claims, wherein the detecting comprises proximity ligation assay or proximity extension assay, and wherein at least one of the plurality of binding agent-oligonucleotide conjugates comprises an binding agent specific for at least one extracellular vesicle-specific marker.

35. The method of the immediately preceding claim, wherein the at least one extracellular vesicle-specific marker comprises one or more of CD81, CD63, CD9, ALIX, CD40, Hsp60, TSG1, or phosphatidylserine.

36. The method of the immediately preceding claim, wherein the digital PCR is droplet digital PCR.

37. The method of any one of the preceding claims, wherein the sequencing comprises next generation sequencing.

38. The method of any one of claims the preceding claims, wherein the sequencing comprises single-cell sequencing.

39. The method of any one of the preceding samples, comprising isolating at least a portion of the extracellular vesicles in the sample from other components of the sample, thereby providing isolated extracellular vesicles.

40. The method of the immediately preceding claim, wherein the isolating comprises affinity purification, immunoprecipitation, size-exclusion chromatography, or centrifugation.

41. The method of any one of claims 39-40, wherein the isolating comprises capturing extracellular vesicles in the sample using one or more extracellular vesicle-specific markers.Atty. Docket No. GH0250WO42. The method of any one of claims 39-41, wherein the isolating comprises capturing extracellular vesicles in the sample using one or more binding agents specific for one or more extracellular vesicle-specific markers.

43. The method of the immediately preceding claim, wherein the one or more binding agents is bound to a solid support.

44. The method of claim 42 or claim 43, wherein the binding agent comprises an antibody, a knottin, an aptamer, an affimer, an avimer, a nanobody, a DARPin, a monobody, or an affinity clamp.

45. The method of any one of claims 42-44, wherein the binding agent comprises an antibody.

46. The method of any one of claims 42-45, wherein the one or more extracellular vesiclespecific markers comprises one or more of CD81, CD63, CD9, ALIX, CD40, Hsp60, TSG1, or phosphatidylserine.

47. The method of any one of claims 39-46, wherein the isolating occurs prior to the contacting.

48. The method of any one of claims 39-47, wherein the isolating occurs prior to the partitioning.

49. The method of any one of claims 39-48, wherein the isolating occurs after the contacting and prior to the partitioning.

50. The method of any one of the preceding claims, comprising detecting one or more extracellular vesicles.

51. The method of the immediately preceding claim, wherein detecting one or more extracellular vesicles comprises:a) contacting the sample with at least one binding agent-oligonucleotide conjugate comprising a binding agent specific for at least one extracellular vesicle-specific marker; andAtty. Docket No. GH0250WO b) before or after the partitioning, detecting a presence, absence, or level of the at least one extracellular vesicle-specific marker by amplifying and / or sequencing an oligonucleotide of the at least one binding agent-oligonucleotide conjugate comprising a binding agent specific for at least one extracellular vesicle-specific marker;thereby detecting the one or more extracellular vesicles in the sample.

52. The method of the immediately preceding claim, wherein the binding agent comprises an antibody, a knottin, an aptamer, an affimer, an avimer, a nanobody, a DARPin, a monobody, or an affinity clamp.

53. The method of claim 51 or claim 52, wherein the binding agent comprises an antibody.

54. The method of any one of claims 51-53, wherein the detecting the presence, absence, or level of the at least one extracellular vesicle-specific marker occurs after the partitioning and simultaneously with the detecting the presence, absence, or level of each of the plurality of EV-associated target molecules.

55. The method of any one of claims 34-52, wherein the at least one extracellular vesiclespecific marker comprises CD81, CD63, CD9, ALIX, CD40, Hsp60, TSG1, or phosphatidylserine.

56. The method of any one of claims 34-55, wherein the sample is contacted with the at least one binding agent-oligonucleotide conjugate comprising a binding agent specific for at least one extracellular vesicle-specific marker (i) before the sample is contacted with the plurality of binding agent-oligonucleotide conjugates, (ii) at the same time that the sample is contacted with the plurality of binding agent-oligonucleotide conjugates, or (iii) after the sample is contacted with the plurality of binding agent-oligonucleotide conjugates and before the partitioning.

57. The method of any one of claims 39-56, wherein the at least a portion of the extracellular vesicles in the sample are isolated from other components of the sample using a different extracellular vesicle-specific marker than the at least one extracellular vesicle-specific marker used for detecting the one or more extracellular vesicles in the sample.Atty. Docket No. GH0250WO 58. The method of any one of the preceding claims, comprising separating extracellular vesicles in the sample that are bound to the one or more binding agents from other components of the sample, thereby providing separated bound extracellular vesicles.

59. The method of the immediately preceding claims, wherein the separating comprises affinity purification, immunoprecipitation, or a pull down assay.

60. The method of any one of claims 58-59, wherein the separating occurs prior to the partitioning, and wherein at least a portion of the separated bound extracellular vesicles is subsequently partitioned.

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

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

63. 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.

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

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

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

67. 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.

68. The method of any one of the preceding claims, wherein at least one of the plurality of EV-associated target molecules is a cell type marker.Atty. Docket No. GH0250WO69. The method of the immediately preceding claim, wherein the cell type marker is a marker for immune cells or solid tissue cells.

70. The method of the immediately preceding claim, wherein the cell type marker is selected from markers for colon, lung, breast, skin, prostate, stomach, pancreas, and liver cell type markers.

71. The method of any one of the preceding claims, wherein at least one of the plurality of EV-associated target molecules is associated with a disease or condition.

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

73. The method of any one of the preceding claims, wherein the plurality of EV-associated target molecules comprises CD147, 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 (CATX), 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, CD81, and / or Ephrin A2.

74. The method of any one of claims 15-73, wherein the plurality of EV-associated target nucleic acids comprises miR-23a, miR-1246, miR-21 let-7a, miR-1229, miR-150, miR- 223, miR-4732-5p, miR-301a, miR-486-5p, miR-6803-5p, let-7b-3p, miR-139-3p, miR-Atty. Docket No. GH0250WO 145-3p, miR-125a-3p, miR-150-5p, IncRNA colorectal neoplasia differentially expressed - h (CRNDE-h), IncRNA breast cancer anti-estrogen resistance 4 (BCAR4), mRNA keratin associated protein 5-4 (KRTAP5-4), mRNA melanoma associated antigen 3 (MAGEA3), IncRNA urothelial cancer associated 1 (UCA1), circRNA homeodomain interacting protein kinase 3 (HIPK3), IncRNA growth arrest specific 5 (GAS5), LNCV6_116109, LNCV6_98390, LNCV6_84003, LNCV6_98602, LNCV_108266, LNCV6_38772, and / or IncRNA colon cancer associated 2 (CCAT2).

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

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

77. The method of any one of claims 75-76, wherein the subject is a human.

78. The method of any one of claims 75-77, wherein the subject has or is at risk of having the disease or condition.

79. The method of any one of claims 75-78, wherein the method comprises analyzing the plurality of EV-associated target molecules in a subsample of the sample or in a second sample obtained from the same subject from which the first sample is obtained.

80. The method of any one of claims 75-79, comprising determining a likelihood that the subject has precancer.

81. The method of any one of claims 75-80, comprising determining a likelihood that the subject has cancer.

82. The method of any one of the preceding claims, wherein the sequencing comprises generating a plurality of sequencing reads, and wherein the method further comprises mapping the plurality of sequence reads to one or more reference sequences to generate mapped sequence reads, and processing the mapped sequence reads to determine the likelihood that the subject has cancer or precancer.Atty. Docket No. GH0250WO83. 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.

84. 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.

85. The method of the immediately preceding claim, 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.

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