Compositions and methods for assaying circulating molecules

Abstract

Provided herein is a method for detecting and quantifying target molecules associated with cell debris.Also provided herein is a method for determining the likelihood that a subject has a disease or condition, such as cancer.Tumor cell-derived proteins, such as cell surface proteins, embedded in cell debris are detected in blood by identifying biomolecular complexes as tumor-derived if they contain both the cell surface protein of interest and a marker specific to dead or dying cells or their fragments, such as cell membrane debris.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 194,789, filed May 28, 2021, which is incorporated by reference herein in its entirety for all purposes.

[0002] FIELD OF THEINVENTION The present disclosure provides compositions and methods for assaying circulating molecules, such as proteins, derived from circulating cell debris. In some embodiments, the circulating proteins derived from cell debris are derived from tumor cells. In some embodiments, the proteins assayed are derived from subjects who have or are suspected of having a disease or disorder, such as cancer. [Background technology]

[0003] Introduction and Abstract Invasive diagnostic procedures, including biopsies, are commonly used to detect or diagnose cancer, ulcers, liver disease, infections, transplant rejection, and other diseases and disorders, where relevant characteristics of cells or tissues derived from a potential site of disease are analyzed. Detection of diseases and disorders based on the analysis of bodily fluids such as blood ("liquid biopsy") is an attractive alternative. Liquid biopsies are non-invasive and in some cases require only a blood draw. However, it has been difficult to develop accurate and sensitive methods for analyzing proteins in liquid biopsy material. This is in part because some of the same proteins released into bodily fluids due to disease are the same proteins normally present in bodily fluids.

[0004] Cell debris is a component of dead cells that may be released into blood or other body fluids following apoptosis, autophagic cell death, necrosis, or other types of cell death. For example, cell death may result in cell membrane fragmentation, resulting in biomolecular complexes that include cell surface proteins. Many types of diseases involve abnormal cell death or altered cell death, including cancer, autoimmune diseases, infectious diseases and sepsis, myocardial infarction, ischemic injury, brain injury, liver disease, and neurodegenerative diseases. Exosomes and other nanoscale vesicles secreted by cells, such as tumor cells, contain proteins and nucleic acids that can also provide important information about the state of the cells from which they originate. Identifying and quantifying circulating molecules, such as proteins, derived from cell debris and exosomes, and distinguishing such molecules from soluble circulating proteins, can provide important information for detecting diseases of interest. Summary of the Invention [Means for solving the problem]

[0005] The methods herein provide an approach to quantify and identify the levels of circulating molecules, such as proteins derived from cell debris. In some embodiments, tumor cell-derived proteins, such as cell surface proteins embedded in cell debris, are detected in blood by identifying biomolecular complexes as tumor-derived if they contain both the cell surface protein of interest and a marker specific for dead or dying cells or fragments thereof, such as cell membrane debris. In some embodiments, the methods herein may be performed in a single step, e.g., by detecting a signal generated by the presence of both a cell surface protein and a component of cell debris, or may be performed sequentially, e.g., by enriching a sample for a marker of cell debris and then assaying a secondary marker, e.g., a protein of interest, in the enriched sample. In certain embodiments, the methods herein facilitate measuring tumor tissue diagnostic markers in blood with improved specificity and / or sensitivity, e.g., by focusing on markers associated with cell debris, and in some embodiments provide an alternative to immunohistochemical assays of biopsied tissue. Applications of the methods herein also include profiling the cell type distribution of dead or dying cells in blood, and identifying circulating protein signatures that can reflect certain disease states.

[0006] The methods herein may include additional steps that can provide information about DNA mutations and modifications, including but not limited to epigenetic mutations and sequence mutations, in the cfDNA or nucleic acids isolated from exosomes. Such methods, including protein analysis and DNA analysis, can provide even more improved information about the likelihood of a particular disease state in a subject.

[0007] The present disclosure aims to meet the need for improved analysis of molecules originating from dead or dying cells, such as tumor cells. Improved detection of cancer markers in blood allows for more accurate detection (diagnosis) of disorders and therefore allows for improved treatment. Thus, the following exemplary embodiments are provided:

[0008] Embodiment 1 is a method for detecting a cell debris-associated target molecule in a sample, comprising: a) contacting the sample or a subsample thereof with at least one binding molecule, thereby generating a complex comprising the at least one binding molecule and cell debris, wherein the at least one binding molecule binds to a cell debris marker, and the cell debris comprises membrane fragments; and b) detecting the presence or level of at least one target molecule associated with the complex. The method includes:

[0009] Embodiment 1.1 is a method according to embodiment 1, in which the sample or a subsample thereof is contacted with a plurality of different binding molecules, which bind to a plurality of cell debris markers or at least one cell debris marker and at least one target molecule.

[0010] Embodiment 1.2 is a method according to any one of the preceding embodiments, wherein the cellular debris comprises plasma membrane fragments.

[0011] Embodiment 1.3 is a method according to any one of the preceding embodiments, wherein the cellular debris comprises endomembrane fragments.

[0012] Embodiment 1.4 is a method according to any one of the preceding embodiments, wherein the cell debris comprises inner plasma membrane fragments.

[0013] Embodiment 2 is the method of any one of the preceding embodiments, wherein the sample is obtained from a subject.

[0014] Embodiment 3 is the method of any one of the preceding embodiments, wherein the sample is a blood sample.

[0015] Embodiment 4 is the method of embodiment 3, wherein the blood sample is a whole blood sample.

[0016] Embodiment 5 is the method of embodiment 3, wherein the blood sample is a plasma sample.

[0017] Embodiment 6 is the method of embodiment 3, wherein the blood sample is a plasma pellet sample or a buffy coat sample.

[0018] Embodiment 7 is the method of any one of the preceding embodiments, wherein at least one binding molecule is a protein, optionally an antibody.

[0019] Embodiment 8 is a method according to any one of the preceding embodiments, wherein the at least one binding molecule binds to a cell debris marker.

[0020] Embodiment 9 is the method of the immediately preceding embodiment, wherein the cell debris marker is an endomembrane marker.

[0021] Embodiment 10 is the method according to any one of embodiments 8 to 9, wherein the cell debris marker is phosphatidylserine or phosphatidylethanolamine.

[0022] Embodiment 11 is the method of any one of embodiments 8 to 10, wherein the cell debris marker is phosphatidylserine.

[0023] Embodiment 12 is the method of the immediately preceding embodiment, wherein at least one binding molecule is an antibody specific for annexin V or phosphatidylserine.

[0024] Embodiment 13 is the method of embodiment 10, wherein the cell debris marker is phosphatidylethanolamine.

[0025] Embodiment 14 is the method of the immediately preceding embodiment, wherein at least one binding molecule is an antibody specific for phosphatidylethanolamine.

[0026] Embodiment 15 is a method according to any one of the preceding embodiments, wherein at least one binding molecule comprises a label or is conjugated to a solid support.

[0027] Embodiment 16 is the method of the immediately preceding embodiment, wherein at least one binding molecule comprises a label, and the method further comprises capturing the at least one binding molecule by binding the label to a solid support.

[0028] Embodiment 17 is the method of embodiment 15, wherein at least one binding molecule is conjugated to a label, and the label comprises a fluorophore, biotin, a peptide, or an oligonucleotide.

[0029] Embodiment 17.1 is the method of embodiment 15, wherein at least one binding molecule is conjugated to an oligonucleotide.

[0030] Embodiment 18 is the method of any one of embodiments 15 to 16, wherein the solid support comprises beads.

[0031] Embodiment 19 is the method of the immediately preceding embodiment, wherein at least one binding molecule is conjugated to a magnetic bead.

[0032] Embodiment 20 is the method of any one of the preceding embodiments, comprising capturing the complex from the sample or subsample prior to the detecting.

[0033] Embodiment 21 is the method of the immediately preceding embodiment, wherein capturing comprises separating components of the sample or subsample that are not bound to the at least one binding molecule from complexes to which the at least one binding molecule is bound.

[0034] Embodiment 22 is the method of any one of embodiments 20-21, wherein the detecting comprises mass spectrometry of the target molecule associated with the complex.

[0035] Embodiment 23 is the method of any one of embodiments 20 to 21, wherein detecting comprises contacting the complex with at least one binding molecule that binds to a target molecule that can associate with the complex.

[0036] Embodiment 24 is the method of the immediately preceding embodiment, wherein at least one binding molecule that binds to the target molecule associated with the complex is an antibody specific for the target molecule.

[0037] Embodiment 25 is the method according to any one of embodiments 23 to 24, wherein at least one binding molecule that binds to the target molecule comprises a label.

[0038] Embodiment 26 is the method of the immediately preceding embodiment, wherein the label is a fluorophore or an oligonucleotide.

[0039] Embodiment 27 is the method of the immediately preceding embodiment, wherein the label is an oligonucleotide.

[0040] Embodiment 28 is the method of the immediately preceding embodiment, wherein the label is an oligonucleotide and the binding molecule that binds to the cell debris marker comprises an oligonucleotide.

[0041] Embodiment 28.1 is the method of any one of embodiments 17, 17.1, 27, or 28, wherein one or more oligonucleotides comprise DNA.

[0042] Embodiment 28.2 is the method of any one of embodiments 17, 17.1, 27, or 28, wherein one or more oligonucleotides comprises RNA.

[0043] Embodiment 28.3 is the method of any one of embodiments 17, 17.1, 27, 28, 28.1, or 28.2, wherein one or more oligonucleotides are at least partially single-stranded.

[0044] Embodiment 28.4 is the method of any one of embodiments 17, 17.1, 27, 28, 28.1, 28.2, or 28.3, wherein one or more oligonucleotides have a length of at least 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotides.

[0045] Embodiment 28.5 is the method of any one of embodiments 17, 17.1, 27, 28, 28.1, 28.2, 28.3, or 28.4, wherein one or more oligonucleotides independently have a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-25, 26-30, 31-40, or 41-50 nucleotides.

[0046] Embodiment 29 is the method of any one of embodiments 28 to 28.5, wherein the detecting comprises a proximity ligation assay or a proximity extension assay.

[0047] Embodiment 29.1 is the method of any one of embodiments 28 to 29, wherein the oligonucleotide comprises a complementary hybridization sequence 3' to the tag (e.g., a molecular barcode that identifies the binding molecule with which the label is associated and, optionally, provides additional information, e.g., to identify the sample and / or pre-enriched fraction being analyzed).

[0048] Embodiment 29.2 is a method according to any one of embodiments 28 to 29.1, wherein when oligonucleotides containing complementary hybridization sequences are in close proximity (e.g., when the binding molecules to which the oligonucleotides are attached are bound to the same piece of cellular debris), the hybridization sequences hybridize to each other and form a substrate for extension by a DNA polymerase.

[0049] Embodiment 29.3 is a method according to embodiment 29.2, in which a substrate for extension by a DNA polymerase is extended to produce an extension product, and the extension product is detected (e.g., by sequencing or qPCR, either of which may follow an amplification and / or library preparation step), thus indicating the presence of a target molecule associated with the cell debris.

[0050] Embodiment 29.4 is the method of any one of embodiments 28 to 29, wherein the oligonucleotide comprises a tag (e.g., a molecular barcode that identifies the binding molecule with which the label is associated and, optionally, provides additional information, e.g., to identify the sample and / or pre-enriched fraction being analyzed).

[0051] Embodiment 29.5 is a method according to any one of embodiments 28-29 or 29.4, in which a ligation template and a ligase are provided that result in ligation of the oligonucleotides to each other to form a ligation product when the oligonucleotides are in close proximity (e.g., when the binding molecules are bound to the same piece of cellular debris).

[0052] Embodiment 29.6 is the method of embodiment 29.5, further comprising amplifying the ligation product.

[0053] Embodiment 29.7 is a method according to embodiment 29.5 or 29.6, further comprising detecting the ligation product or an amplification product thereof (e.g., by sequencing or qPCR, which may follow the amplification and / or library preparation steps), thus indicating the presence of a target molecule associated with the cell debris.

[0054] Embodiment 30 is the method of any one of embodiments 20 to 29.7, wherein the detecting comprises an immunoassay.

[0055] Embodiment 31 is the method of the immediately preceding embodiment, wherein the immunoassay is an enzyme-linked immunosorbent assay, a sandwich assay, an electrochemiluminescence assay, or a multiplex immunoassay.

[0056] Embodiment 32 is the method of embodiments 20 to 31, wherein the detecting comprises flow cytometric analysis of the complex.

[0057] Embodiment 33 is the method of any one of the preceding embodiments, wherein multiple target molecules associated with the complex are detected.

[0058] Embodiment 34 is the method of the immediately preceding embodiment, wherein the plurality of target molecules is between 2 and 10,000, between 2 and 5,000, between 2 and 1,000, or between 2 and 100 target molecules.

[0059] Embodiment 35 is a method according to any one of embodiments 1 to 19 or 33 to 34, comprising contacting the complex with at least one binding molecule that binds to a target molecule that may be associated with the complex, and then capturing the complex.

[0060] Embodiment 35.1 is a method according to any one of embodiments 1 to 19 or 33 to 34, wherein prior to contacting the sample with the cell debris marker binding molecule, the sample is contacted with at least one binding molecule that binds to the target molecule.

[0061] Embodiment 35.2 is a method according to any one of embodiments 1 to 19 or 33 to 34, wherein after contacting the sample with the cell debris marker binding molecule, the sample is contacted with at least one binding molecule that binds to the target molecule.

[0062] Embodiment 35.3 is a method according to any one of embodiments 1 to 19 or 33 to 34, wherein the sample is contacted with at least one binding molecule that binds to the target molecule simultaneously with contacting the sample with the cell debris marker binding molecule.

[0063] Embodiment 36 is the method of any one of the preceding embodiments, wherein at least one of the target molecules, two or more of the plurality of target molecules, or each of the plurality of target molecules is a protein.

[0064] Embodiment 37 is a method according to any one of the preceding embodiments, wherein at least one target molecule, two or more of the plurality of target molecules, or each of the plurality of target molecules is a carbohydrate, optionally a glycoprotein carbohydrate.

[0065] Embodiment 38 is the method of any one of the preceding embodiments, wherein at least one target molecule is a molecule associated with a disease, two or more of the plurality of target molecules are molecules associated with a disease, or each of the plurality of target molecules is a molecule associated with a disease.

[0066] Embodiment 39 is the method of the immediately preceding embodiment, wherein the disease is cancer.

[0067] Embodiment 40 is a method according to the immediately preceding embodiment, wherein at least one target molecule is upregulated in tumor cells relative to healthy cells of the same tissue type.

[0068] Embodiment 41 is the method of any one of embodiments 38 to 40, wherein at least one, two or more, or each of the target molecules is selected from PD-L1, CTLA4, NYESO1, mesothelin, CA15-3, CA19-9, CA-125, and CA-172-4.

[0069] Embodiment 42 is the method of any one of the preceding embodiments, wherein at least one target molecule, two or more target molecules, or each of the multiple target molecules is a cell type marker.

[0070] Embodiment 43 is the method of the immediately preceding embodiment, wherein the cell type marker is selected from a marker for an immune cell and a solid tissue cell.

[0071] Embodiment 44 is the method of the immediately preceding embodiment, wherein the cell type markers are selected from markers of colon, lung, breast, skin, prostate, stomach, pancreas, and liver cell type markers.

[0072] Embodiment 45 is the method of any one of the preceding embodiments, wherein the sample is obtained from a subject having a disease, and wherein the detecting comprises identifying a plurality of target molecules, and wherein the identifying comprises mass spectrometry of the target proteins.

[0073] Embodiment 46 is a method according to any one of the preceding embodiments, comprising measuring the total cell debris level in the sample or a subsample thereof.

[0074] Embodiment 46.1 is a method according to the immediately preceding embodiment, wherein the total cell debris level is measured by quantifying at least one cell debris marker in the sample or a subsample thereof.

[0075] Embodiment 47 is a method according to embodiment 46 or 46.1, wherein the sample is obtained from a subject having a disease and the total cell debris level is measured relative to the total cell debris level in a sample or a subsample thereof obtained from a healthy individual.

[0076] Embodiment 48 is the method of any one of the preceding embodiments, comprising analyzing DNA in a subsample of the sample, or in a second sample obtained from the same subject from which the first sample was obtained.

[0077] Embodiment 49 is the method of the immediately preceding embodiment, wherein the aliquot or second sample is a plasma or serum sample.

[0078] Embodiment 50 is the method of the immediately preceding embodiment, wherein the DNA is cfDNA.

[0079] Embodiment 50.1 is a method according to any one of embodiments 48 to 50, wherein the analysis of the DNA includes quantifying at least one epigenetic feature of the target region of the DNA, and optionally, the epigenetic feature includes methylation.

[0080] Embodiment 50.2 is the method of any one of embodiments 48 to 50, wherein analyzing the DNA comprises detecting or quantifying one or more genetic variants in one or more target regions of the DNA.

[0081] Embodiment 51 is a method for detecting the presence or absence of cancer, comprising performing a method according to any one of the preceding embodiments, wherein the presence or level of at least one target molecule associated with the complex indicates the presence or absence of cancer.

[0082] Embodiment 52 is a method for screening for cancer, comprising carrying out the method of any one of embodiments 1 to 50.2 on samples from a plurality of subjects, wherein the presence or level of at least one target molecule associated with a complex indicates that the corresponding subject may have cancer.

[0083] Embodiment 53 is a method for monitoring residual cancer or detecting the presence or absence of recurrent cancer, comprising carrying out a method according to any one of embodiments 1 to 50.2, wherein the presence or level of at least one target molecule associated with the complex indicates the cancer status or the presence or absence of recurrent cancer.

[0084] Embodiment 54 is a method for identifying a therapy for treating a disease, optionally wherein the disease is cancer, the method comprising carrying out a method according to any one of embodiments 1 to 50.2, wherein the presence or level of at least one target molecule associated with the complex indicates a suitable therapy for treating the disease. [Brief description of the drawings]

[0085] [Figure 1A] 1A-1B show an exemplary workflow of the methods disclosed herein. At least a portion of a whole blood sample is fractionated into plasma, buffy coat, and red blood cells. The plasma is fractionated into a plasma pellet and a plasma supernatant. In FIG. 1A, cell debris is isolated from the buffy coat fraction and the plasma pellet, and / or from a portion of the whole blood sample. Also, exosomes can be isolated from the plasma supernatant and / or from a portion of the whole blood sample. Target proteins associated with the cell debris and / or exosomes are detected. In another exemplary method shown in FIG. 1B, the buffy coat fraction, the plasma pellet, the plasma supernatant, and / or a portion of the whole blood sample are contacted with an antibody specific for a marker or target protein that is linked to an oligonucleotide that facilitates hybridization and extension when the marker and the target protein are in close proximity, and subsequent chain extension, amplification, and sequencing to detect the target protein. In another embodiment, the oligonucleotides can be configured to facilitate ligation when the marker and target protein are in close proximity, followed by chain extension, amplification, and sequencing to detect the target protein. [Figure 1B]1A-1B show an exemplary workflow of the methods disclosed herein. At least a portion of a whole blood sample is fractionated into plasma, buffy coat, and red blood cells. The plasma is fractionated into a plasma pellet and a plasma supernatant. In FIG. 1A, cell debris is isolated from the buffy coat fraction and the plasma pellet, and / or from a portion of the whole blood sample. Also, exosomes can be isolated from the plasma supernatant and / or from a portion of the whole blood sample. Target proteins associated with the cell debris and / or exosomes are detected. In another exemplary method shown in FIG. 1B, the buffy coat fraction, the plasma pellet, the plasma supernatant, and / or a portion of the whole blood sample are contacted with an antibody specific for a marker or target protein that is linked to an oligonucleotide that facilitates hybridization and extension when the marker and the target protein are in close proximity, and subsequent chain extension, amplification, and sequencing to detect the target protein. In another embodiment, the oligonucleotides can be configured to facilitate ligation when the marker and target protein are in close proximity, followed by chain extension, amplification, and sequencing to detect the target protein.

[0086] [Diagram 2] FIG. 2 is a schematic diagram of one example of a system suitable for use in some embodiments of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0087] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS Reference will now be made in detail to certain specific embodiments of the invention. While the invention will be described in conjunction with such embodiments, it will be understood that it is not intended to limit the invention to such embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents which may be included within the invention as defined by the appended claims.

[0088] Before describing the present teachings in detail, it should be understood that the present disclosure is not limited to specific compositions or process steps, which may vary as such.As used herein and in the appended claims, it should be noted that the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise.Thus, for example, a reference to "nucleic acid" includes a plurality of nucleic acids, a reference to "cell" includes a plurality of cells, and so on.

[0089] Numeric ranges are inclusive of the numbers defining the range. Measurements and measurable values ​​are understood to be approximations taking into account significant digits and error associated with measurement. Additionally, 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 and detailed description are exemplary and explanatory only and are not restrictive of the teachings.

[0090] Unless otherwise noted in the specification above, embodiments herein that are described as "comprising" various components are also contemplated as "consisting of" or "consisting essentially of" the described components, and embodiments herein that are described as "consisting of" various components are also contemplated as "comprising" or "consisting essentially of" the described components, and embodiments herein that are described as "consisting essentially of" various components are also contemplated as "consisting of" or "comprising" the described components (this synonymy does not apply to the use of such terms in the claims).

[0091] The section headings used herein are for organizational purposes and should not be construed as limiting the subject matter of this disclosure in any way. In the event that any document or other material incorporated by reference conflicts with any express content of this specification, including definitions, the present specification shall control. I. Definition

[0092] "Cell debris marker" as used herein refers to a molecule, such as a protein, lipid, or carbohydrate, that is physically associated with or embedded in a component of a dead or dying cell and is present in a greater proportion in such a component of a disrupted or intact dead or dying cell than on the outer membrane of an intact live cell, in an intact vesicle, or in the soluble fraction of a sample. The component of a dead or dying cell that is associated with a cell debris marker may be dissociated from other components of the cell from which it originated, or may be contained in an intact dead or dying cell. Examples of cell debris markers include, but are not limited to, molecules that are associated with or localized to the inner plasma membrane, such as phosphatidylserine and phosphatidylethanolamine.

[0093] A "dying cell," as used herein, is an intact pre-apoptotic, pre-necrotic, or autophagic cell in which physical changes associated with cell death have begun to result, for example, in the translocation of internal phospholipids to the outside of the plasma membrane.

[0094] "Cell debris" as used herein refers to components of dead cells that may be released into blood or other bodily fluids following apoptosis, autophagic cell death, necrosis, or other types of cell death. For example, cell death may be coupled to cell membrane fragmentation, resulting in biomolecular complexes that include cell surface proteins. In some embodiments, cell debris includes membrane fragments released from dead or dying cells, as well as associated molecules such as proteins and / or carbohydrates.

[0095] "Cell debris marker binding molecule" and "binding molecule" that "binds to cell debris marker" as used herein refer to a molecule that specifically binds to a cell debris marker. For example, an antibody that specifically binds to a cell debris marker is a cell debris marker binding molecule. Examples of cell debris marker binding molecules include, but are not limited to, annexin V, antibodies against phosphatidylserine, and antibodies against phosphatidylethanolamine. Binding molecules also include nanobodies, aptamers, affimers, and DARPins.

[0096] A first molecule is "associated with" a complex or other molecule when the first molecule is bound to the complex or other molecule directly or indirectly (e.g., through the chains of one or more additional molecules).

[0097] "Exosome marker" as used herein refers to a molecule, such as a protein, lipid, or carbohydrate, that is physically associated with or embedded in the outer membrane of an exosome and is present in a higher proportion in exosomes than on the outer membrane of an intact live cell, in cell debris, or in the soluble fraction of a sample. Examples of exosome markers include, but are not limited to, tetraspanins, CD9, CD63, and CD81.

[0098] "Exosome marker binding molecule" as used herein means a molecule that specifically binds to an exosome marker. For example, an antibody that specifically binds to an exosome marker is an exosome marker binding molecule. Binding molecules also include nanobodies, aptamers, affimers, and DARPins.

[0099] "Cell type marker," as used herein, means a molecule that is present in a higher proportion of one or more cell types than other cell types present in the same sample, or than any other cell types.

[0100] "Solid tissue cells" as used herein refers to cells in or derived from solid tissue. Solid tissue cells exclude circulating cell types, such as cells normally found in blood or lymph. Examples of solid tissue cell types include, but are not limited to, colon, lung, breast, skin, prostate, stomach, pancreas, and liver cells.

[0101] "Cell-free DNA", "cfDNA molecules", or simply "cfDNA" includes DNA molecules that are naturally present in a subject in an extracellular form (e.g., in blood, serum, plasma, or other bodily fluids such as lymph, cerebrospinal fluid, urine, or sputum). cfDNA was previously present in one or more cells of a large, complex biological organism, e.g., a mammal, but has been released from the cell(s) into fluids found within the organism, and can be obtained from a sample of the fluid, without the need to perform an in vitro cell lysis step. cfDNA molecules may occur as DNA fragments.

[0102] As used herein, a "blood sample" refers to a sample containing whole blood or a component thereof (eg, plasma, serum, buffy coat, plasma pellet).

[0103] As used herein, "partitioning" of nucleic acids, such as DNA molecules, refers to separating, fractionating, sorting, or enriching a sample or population of nucleic acids into multiple subsamples or subpopulations of nucleic acids based on one or more modifications or features that are present in different proportions in each of the multiple subsamples or subpopulations. Partitioning may include physically distributing nucleic acid molecules based on the presence or absence of one or more methylated nucleic acid bases. A sample or population can be distributed into one or more distributed subsamples or subpopulations based on a trait that indicates a genetic or epigenetic change or a disease state.

[0104] As used herein, the form of a sample "originally isolated" refers to the composition or chemical structure of the sample at the time the sample is isolated and before it is subjected to any procedure that alters the chemical structure of the isolated sample. Similarly, a feature "originally present" in a molecule refers to a feature that is present in the "original molecule" or in a molecule that "originally comprises" the feature before the molecule is subjected to any procedure that alters the chemical structure of the molecule.

[0105] As used herein, "base pairing specificity" refers to the standard DNA base (A, C, G, or T) with which a given base most preferentially pairs. For example, unmodified cytosine and 5-methylcytosine have the same base pairing specificity (i.e., specificity for G), whereas uracil and cytosine have different base pairing specificities, since uracil has base pairing specificity for A and cytosine has base pairing specificity for G. Nevertheless, uracil's ability to form a wobble pair with G is irrelevant, since uracil most preferentially pairs with A among the four standard DNA bases.

[0106] As used herein, a "combination" containing multiple members refers to either a single composition containing the members, or a set of compositions that are in close proximity, for example, in separate containers or compartments of a larger container, such as a multi-well plate, tube rack, refrigerator, freezer, incubator, water bath, ice bucket, machine, or other form of storage.

[0107] "Capture" of one or more target molecules, such as one or more proteins or nucleic acids or one or more molecules that comprise at least one target region, refers to preferentially isolating or separating the one or more target molecules from non-target molecules.

[0108] As used herein, a "label" is a capture moiety, fluorophore, oligonucleotide, or other moiety that facilitates detection, separation, or isolation of the one to which it is attached.

[0109] As used herein, a "capture moiety" is a molecule that allows for affinity separation of a molecule linked to the capture moiety from a molecule that lacks the capture moiety. Exemplary capture moieties include biotin, which allows for affinity separation by binding to streptavidin that is linked or linkable to a solid phase, or oligonucleotides, which allow for affinity separation by binding to complementary oligonucleotides that are linked or linkable to a solid phase.

[0110] As used herein, a "tag" is a molecule, such as a nucleic acid, a label, a fluorophore, or a peptide, that contains information that indicates the characteristics of the molecule with which it is associated. For example, a molecule may have a sample tag (distinguishing a molecule in one sample from a molecule in another sample), a molecular tag / molecular barcode / barcode (distinguishing different molecules from each other (in both unique and non-unique tagging scenarios), a partitioning tag (distinguishing a molecule in a partitioning fraction from a molecule in another partitioning fraction), a purification tag, and / or a detectable tag or label.

[0111] As used herein, a "target molecule" is a molecule, such as a protein, carbohydrate, or lipid, whose presence or absence is detected. The identity of the target molecule does not need to be known prior to detection. The identity of the target molecule may be determined as part of the method described herein, for example, by analyzing the target protein using mass spectrometry.

[0112] "Specifically binds," in the context of a primer, probe, or other oligonucleotide, protein, or other binding molecule and a target sequence, means that under appropriate hybridization conditions, the primer, oligonucleotide, or probe hybridizes to its target sequence or a copy thereof to form a stable hybrid, while minimizing the formation of stable non-target hybrids. Thus, the primer or probe hybridizes to the target sequence or a copy thereof to a sufficiently greater extent than to the non-target sequence, ultimately allowing capture or detection of the target sequence. Suitable hybridization conditions are well known in the art and can be predicted based on sequence composition or can be determined using routine testing methods (see, e.g., sections 1.90-1.91, 7.37-7.57, 9.47-9.51, and 11.47-11.57 of Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), particularly sections 9.50-9.51, 11.12-11.13, 11.45-11.47, and 11.55-11.57, which are incorporated herein by reference).

[0113] "Plasma pellet" as used herein means the precipitated material after centrifugation of plasma that has previously been separated from whole blood. Plasma can be separated from whole blood by a first centrifugation, and the plasma pellet can be generated by a second centrifugation of only the plasma portion (supernatant) of the first centrifugation. The supernatant of the second centrifugation may be referred to as isolated plasma, and the precipitate is the plasma pellet. In some embodiments, the plasma pellet contains cellular debris (e.g., generally lower in mass or smaller in size than the cellular debris found in the buffy coat). In some embodiments, the plasma pellet is substantially free of cfDNA, cfRNA, soluble proteins, exosomes, and metabolites. "Substantially free" means absent to a sufficient degree that the relevant properties are not meaningfully affected by the presence of small amounts of impurities.

[0114] "Immunoassay," as used herein, refers to an assay or method that involves contacting a molecule or sample with an antibody to test a function or to detect the presence, identify, and / or quantify one or more components of the sample. Examples of immunoassays can include, but are not limited to, enzyme-linked immunosorbent assays (ELISAs), sandwich assays, electrochemiluminescence (ECL) assays, and multiplex assays.

[0115] "Antibody", as used herein, is used broadly to encompass a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0116] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody and binds to an 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.

[0117] A protein or nucleic acid is said to be "produced by a tumor" if it originates from a tumor cell. DNA that originates from a tumor cell is "circulating tumor DNA" ("ctDNA"). Tumor cells are neoplastic cells that originate from a tumor, whether they remain in the tumor or are detached from the tumor (e.g., as in the case of metastatic cancer cells and circulating tumor cells).

[0118] A "target region" in the context of a nucleic acid refers to a genomic locus that is targeted for identification and / or capture, e.g., by use of a probe (e.g., by sequence complementarity). A "target region set" or "set of target regions" refers to a plurality of genomic loci that are targeted for identification and / or capture, e.g., by use of a set of probes (e.g., by sequence complementarity).

[0119] "Sequence variable target region" refers to a target region that may exhibit sequence changes, such as nucleotide substitutions (i.e., single base mutations), insertions, deletions, or gene fusions or translocations, in neoplastic cells (e.g., tumor cells and cancer cells) compared to normal cells. A sequence variable target region set is a set of sequence variable target regions. In some embodiments, a sequence variable target region is a target region that may exhibit changes affecting less than or equal to 50 consecutive nucleotides, such as less than or equal to 40, 30, 20, 10, 5, 4, 3, or 2 nucleotides, or affecting one nucleotide.

[0120] "Epigenetic target region" refers to a target region that may show sequence-independent differences in different cell or tissue types (e.g., different types of immune cells) or neoplastic cells (e.g., tumor cells and cancer cells) compared to normal cells, or in DNA such as cfDNA from a different cell type or subject with cancer compared to DNA such as cfDNA from a healthy subject, or in cfDNA originating from a different cell or tissue type (e.g., immune, lung, colon, etc.) that does not normally contribute substantially to cfDNA compared to background cfDNA (e.g., cfDNA originating from hematopoietic cells). Examples of sequence-independent changes include, but are not limited to, changes in methylation (increase or decrease), nucleosome distribution, cfDNA fragmentation patterns, CCCTC-binding factor ("CTCF") binding, transcription start sites, and regulatory protein binding regions. Thus, epigenetic target region sets include, but are not limited to, hypermethylated variable target region sets, hypomethylated variable target region sets, and fragmented variable target region sets such as CTCF binding sites and transcription start sites. For the present purpose, epigenetic target region set can also include the loci that are susceptible to local amplification and / or gene fusion associated with neoplasm, tumor or cancer.Because the detection of copy number changes by sequencing or the detection of fusion sequences that map to more than one locus in a reference genome tends to be more similar to the detection of the exemplary epigenetic changes discussed above than the detection of nucleotide substitutions, insertions or deletions, for example, in that local amplifications and / or gene fusions can be detected with relatively shallow depth sequencing, since their detection does not depend on the accuracy of base calls at one or a few individual positions.Epigenetic target region set is a set of epigenetic target regions.

[0121] The "capture yield" of a collection of probes for a given target set refers to the amount of nucleic acid corresponding to the target set that the collection of probes captures under typical conditions (e.g., the amount relative to another target set or the absolute amount). Exemplary typical capture conditions are incubation of sample nucleic acid and probes at 65°C for 10-18 hours in a small reaction volume (approximately 20 μL) containing stringent hybridization buffer. Capture yields may be expressed as absolute values ​​or, in the case of a collection of multiple probes, as relative values. When comparing capture yields of multiple sets of target regions, capture yields are normalized to the footprint size of the set of target regions (e.g., on a per kilobase basis). Thus, for example, when the footprint sizes of the first and second target regions are 50 kb and 500 kb, respectively (the normalization factor is 0.1), the DNA corresponding to the first set of target regions is captured with a higher yield than the DNA corresponding to the second set of target regions when the mass concentration per volume of the captured DNA corresponding to the first set of target regions is greater than 0.1 times the mass concentration per volume of the captured DNA corresponding to the second set of target regions. As a further example, using the same footprint size, the DNA corresponding to the first set of target regions is captured with a capture yield that is 2 times greater than the DNA corresponding to the second set of target regions when the captured DNA corresponding to the first set of target regions has a mass concentration per volume that is 0.2 times the mass concentration per volume of the captured DNA corresponding to the second set of target regions.

[0122] The term "methylation" or "DNA methylation" refers to the addition of a methyl group to a nucleobase of a nucleic acid molecule. In some embodiments, methylation refers to the addition of a methyl group to a cytosine at a CpG site (cytosine-phosphate-guanine site (i.e., a cytosine followed by a guanine in the 5'->3' direction of a nucleic acid sequence). In some embodiments, DNA methylation refers to the addition of a methyl group to a cytosine at a CpG site (cytosine-phosphate-guanine site (i.e., a cytosine followed by a guanine in the 5'->3' direction of a nucleic acid sequence). 6-methyladenine, etc. In some embodiments, DNA methylation is 5-methylation (modification of the carbon at the 5th position of the 6-membered carbon ring of cytosine). In some embodiments, 5-methylation refers to the addition of a methyl group to the 5C position of cytosine to create 5-methylcytosine (5mC). In some embodiments, methylation includes derivatives of 5mC. Derivatives of 5mC include, but are not limited to, 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), and 5-caryboxylcytosine (5-caC). In some embodiments, DNA methylation is 3C methylation (modification of the carbon at the 3rd position of the 6-membered carbon ring of cytosine). In some embodiments, 3C methylation includes the addition of a methyl group to the 3C position of cytosine to create 3-methylcytosine (3mC). Methylation can also occur at non-CpG sites, for example, methylation can occur at CpA, CpT, or CpC sites. DNA methylation can alter the activity of methylated DNA regions. For example, when DNA in a promoter region is methylated, gene transcription can be repressed. DNA methylation is important for normal development, and aberrant methylation can disrupt epigenetic regulation. Disruption of epigenetic regulation, for example repression, can cause diseases such as cancer. Promoter methylation of DNA can indicate cancer.

[0123] The term "hypermethylated" refers to an increased level or degree of methylation of a nucleic acid molecule(s) relative to other nucleic acid molecules within a population of nucleic acid molecules (e.g., a sample). In some embodiments, hypermethylated DNA may include DNA molecules that contain at least one methylated residue, at least two methylated residues, at least three methylated residues, at least five methylated residues, or at least ten methylated residues.

[0124] The term "hypomethylated" refers to a decreased level or degree of methylation of a nucleic acid molecule(s) relative to other nucleic acid molecules in a population (e.g., a sample) of nucleic acid molecules. In some embodiments, hypomethylated DNA includes unmethylated DNA molecules. In some embodiments, hypomethylated DNA may include DNA molecules that include 0 methylated residues, at most 1 methylated residue, at most 2 methylated residues, at most 3 methylated residues, at most 4 methylated residues, or at most 5 methylated residues.

[0125] The term "agent that recognizes modified nucleobases of DNA", e.g., "agent that recognizes modified cytosines of DNA", refers to a molecule or reagent that binds to or detects one or more modified nucleobases of DNA, e.g., methylcytosines. A "modified nucleobase" is a nucleobase that includes a difference in chemical structure from an unmodified nucleobase. In the case of DNA, the unmodified nucleobase is adenine, cytosine, guanine, or thymine. In some embodiments, the modified nucleobase is a modified cytosine. In some embodiments, the modified nucleobase is a methylated nucleobase. In some embodiments, the modified cytosine is a methylcytosine, e.g., 5-methylcytosine. In such embodiments, the cytosine modification is methyl. Agents that recognize methylcytosines of DNA include, but are not limited to, "methyl-binding reagents", which herein refer to reagents that bind to methylcytosines. Methyl-binding reagents include, but are not limited to, methyl-binding domains (MBDs), methyl-binding proteins (MBPs), and antibodies specific for methylcytosines. In some embodiments, such antibodies bind to 5-methylcytosines of DNA. In some such embodiments, the DNA may be single-stranded or double-stranded.

[0126] The terms "or combinations thereof" and "or combinations thereof" as used herein refer to any and all permutations and combinations of the terms listed before 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 the particular context, BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing with this example, combinations including repeats of one or more items or terms are explicitly included, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, and CABABB. Those skilled in the art will understand that there is typically no limit to the number of items or terms in any combination, unless otherwise clear from the context.

[0127] "Or" is used in its inclusive sense, i.e., equivalent to "and / or," unless the context requires otherwise. II. Exemplary Methods A. Identification and quantification of cell debris-associated target molecules

[0128] The method disclosed herein includes contacting a sample or a subsample with at least one cell debris marker binding molecule and detecting the presence or level of at least one target molecule, such as a target protein, associated with the cell debris. The cell debris includes membrane fragments. The target molecule is different from the cell debris marker. The detection of the target molecule associated with the cell debris may be more beneficial than detecting the target molecule in, for example, a whole blood or plasma sample, because the latter may show a higher background or baseline level of the target protein, whereas the level of the target molecule associated with the cell debris is low if the subject is healthy. In some embodiments, the at least one target molecule, such as a target protein, associated with the cell debris is quantified. In some embodiments, the post-translational modification of the target protein associated with the cell debris is detected or quantified.

[0129] As shown in FIG. 1A, cell debris can be enriched or isolated from a whole blood sample or various subsamples thereof prepared, for example, by centrifugation. In some embodiments, one or more or each of the whole blood sample, the buffy coat fraction, and the plasma pellet are contacted with at least one cell debris marker binding molecule. An exemplary cell debris marker binding molecule is Annexin V or its conjugate, for example, with biotin. Other exemplary cell debris marker binding molecules are described elsewhere herein. One or more target molecules, such as proteins, can be detected or quantified in the enriched cell debris, which may include apoptotic bodies. In some embodiments, exosomes are also enriched from the sample or a subsample thereof, for example, from the supernatant after a second centrifugation of plasma. The one or more target molecules, which may be the same or different as the one or more target molecules detected or quantified in the enriched cell debris, if applicable, can be detected or quantified in the enriched exosomes. In some embodiments, cell-free DNA is isolated from the sample or a subsample thereof, for example, from the supernatant after a second centrifugation of plasma. The cell-free DNA can be analyzed, for example, as described elsewhere herein, to detect or quantitate sequence variations or epigenetic signatures.

[0130] The contacting and detecting can be performed sequentially or simultaneously. In some embodiments, the sequential method includes enriching, capturing, or isolating a complex comprising at least one cell debris marker binding molecule, at least one cell debris marker, and one or more target molecules, and then detecting one or more target molecules. In some embodiments, the method includes simultaneously contacting a sample or a subsample with at least one cell debris marker binding molecule and at least one binding molecule specific to the target molecule, the binding molecule being configured to facilitate direct detection of the target molecule associated with the cell debris, such as a proximity ligation assay or a proximity extension assay. See, for example, the proximity ligation assay shown in FIG. 1B. For example, FIG. 1B shows the use of oligo-linked antibodies or conjugates (e.g., antibodies specific to one or both of PDL1 and CTLA4, and annexin V, or antibodies specific to phosphatidylserine) to detect or quantify PDL1 and / or CTLA4 in cell debris. If desired, exosomes may be analyzed using similar proximity ligation or proximity extension techniques with plasma supernatant and / or cell-free DNA may be analyzed as discussed above with respect to FIG. 1A.

[0131] In a proximity extension assay, the first and second binding molecules (e.g., cell debris marker binding molecules and target binding molecules) are labeled with an oligonucleotide that includes a complementary hybridization sequence that is 3' to a tag (e.g., a molecular barcode that identifies the binding molecule to which the label is associated; the tag further includes one or more additional elements that provide additional information related to the sample, e.g., a sample tag, and / or related to the pre-enriched fraction being analyzed, e.g., a partitioning tag; this can facilitate subsequent pooling). The tag may have any of the characteristics described elsewhere herein for tags. When the oligonucleotides are in proximity (e.g., as occurs when the binding molecules are bound to the same cell debris piece), the hybridization sequences can hybridize to each other and form a substrate for extension by DNA polymerase at a rate above background. The extension product can then be detected (e.g., by sequencing or qPCR, which may be followed by an amplification and / or library preparation step), thus indicating the presence of the target molecule associated with the cell debris. For a general example of proximity extension assay, see Anderson et al., WO2007 / 005649A2, which is incorporated herein by reference for all purposes.In addition, amplification of nucleic acid labels attached to antibodies is also discussed in Sano et al., US5,665,539, which is incorporated herein by reference for all purposes.

[0132] In a proximity ligation assay, the first and second binding molecules (e.g., cell debris marker binding molecule and target binding molecule) are labeled with oligonucleotides. A ligation template and a ligase are provided to effect ligation of the oligonucleotides to each other at a rate above background when they are in close proximity (e.g., occurs when the binding molecules are bound to the same cell debris piece). The ligation product may comprise at least a portion of the ligation template and / or may be at least partially double-stranded, for example, by hybridization of a portion of the ligation template and / or a complementary strand synthesis step using an appropriate primer. The oligonucleotide may comprise a tag and / or a barcode as described above and elsewhere herein. The tag may have any of the characteristics described elsewhere herein for tags. The ligation product may be a substrate for amplification. The ligation product can be detected (e.g., by sequencing or qPCR, which may be followed by an amplification and / or library preparation step), thus indicating the presence of a target molecule associated with the cell debris. General examples of proximity ligation assays can be found in US 9,518,296 B2 to Ruff et al., and US 2007 / 0281367 A1 to Hennessy et al., both of which are incorporated herein by reference for all purposes.

[0133] Also disclosed are methods in which exosome marker-binding molecules are used in place of or in combination with cell debris marker-binding molecules, which may include enriching, capturing, or isolating a complex comprising at least one exosome marker-binding molecule, at least one exosome marker, and one or more target molecules, and then detecting, for example, one or more target molecules derived from exosomes in addition to the one or more target molecules in the complex comprising the cell debris marker. Exosomes are not considered cell debris.

[0134] In some embodiments, such methods include contacting a sample or a subsample with a cell debris marker binding molecule and detecting one or more target molecules, such as target proteins, associated with the cell debris. In some embodiments, the cell debris marker is an inner membrane marker. Without wishing to be bound by theory, lipids that are almost exclusively localized in the inner plasma membrane leaflet are inverted to the outer membrane leaflet in pre-apoptotic cells, apoptotic cells, and other dying and dead cells. In addition, disrupted cellular components include the inner membrane leaflet that is exposed to the sample solvent. Thus, selectively binding inner membrane markers can facilitate selective binding to cell debris and thus selectively detect target molecules associated with cell debris over soluble proteins. Exemplary inner membrane markers include, but are not limited to, phosphatidylserine and phosphatidylethanolamine. In some embodiments, the cell debris marker binding molecule is a protein, such as an antibody, nanobody, affimer, or DARpin, that specifically binds to the cell debris marker. In some embodiments, the protein is annexin V, or an antibody specific for phosphatidylserine. In some embodiments, the cell debris marker binding molecule is a nucleic acid, such as an aptamer. In some embodiments, the cell debris marker binding molecule comprises a label, such as a capture moiety (e.g., biotin) or an oligonucleotide.

[0135] In some embodiments, such methods include measuring the total cell debris level in a sample or a subsample thereof. The total cell debris level can be measured, for example, by quantifying the total amount of cell debris markers, which can be any of those described herein. The cell debris marker used to measure the total cell debris level can be the same as or different from the cell debris marker that the binding molecule binds to. If more than one cell debris marker binding molecule is used, the cell debris marker used to measure the total cell debris level can be the same as one of the cell debris markers that the binding molecule binds to, or can be different from all of the cell debris markers that the binding molecule binds to. Any suitable measurement technique can be used for such measurement (e.g., immunoassay, mass spectrometry, etc.).

[0136] The methods herein can also be used to assay exosomes. In such embodiments, such methods include contacting a sample or a subsample with at least one exosome marker binding molecule and detecting the presence or level of at least one target molecule, such as a target protein, associated with an exosome. In some embodiments, the exosome marker is a transmembrane protein that is present in a higher proportion in the exosome membrane than in other membranes in the sample. In some embodiments, the exosome marker is a tetraspanin. In some embodiments, the exosome marker is CD9, CD63, or CD81. In some embodiments, the exosome marker binding molecule is a protein, such as an antibody, nanobody, affimer, or DARpin, that specifically binds to the exosome marker. In some embodiments, the protein is an antibody specific for CD9, CD63, or CD81. In some embodiments, the exosome marker binding molecule is a nucleic acid, such as an aptamer. In some embodiments, the exosome marker binding molecule includes a label or capture moiety, such as biotin.

[0137] The methods herein include detecting at least one target molecule, such as a target protein. In some embodiments, the identity of one or more target molecules is unknown before the initiation of the method. In some such embodiments, such methods include detecting the target protein using mass spectrometry and target protein identification. In some embodiments, the detection includes contacting the sample with a binding molecule specific for the target molecule suspected to be present in the sample. In some embodiments, the identity of one or more target molecules is known before the initiation of the method, and the target molecule detection method is selected accordingly. In some embodiments, the detection includes performing an immunoassay, such as an ELISA, a sandwich assay, an electrochemiluminescence (ECL) assay, or a multiplex immunoassay. In some embodiments, the detection includes flow cytometric analysis of the sample.

[0138] In some embodiments, the one or more target molecules are molecules derived from tumor cells, cells in another disease state, or cells altered by the presence of disease in the subject from which the cells are obtained. In some embodiments, the one or more target molecules are derived from a cell type that is not normally present in the type of body sample obtained from the subject. In some embodiments, at least one target molecule is a target protein. In some embodiments, at least one target molecule is a target carbohydrate, such as a glycoprotein carbohydrate. In some embodiments, the one or more target molecules are selected from PD-L1, CTLA4, NYESO1, mesothelin, CA15-3, CA19-9, CA-125, and CA-172-4. In some embodiments, the one or more target molecules are cell type markers, such as immune cell type markers or solid tissue cell type markers. In some embodiments, the solid tissue cell type markers are markers present in colon, lung, breast, skin, prostate, stomach, pancreas, or liver cells.

[0139] In some embodiments, determining the level of the target molecule facilitates disease diagnosis or identification of appropriate treatment. In some embodiments, the presence or alteration of the level of one or more target molecules indicates the presence of a disease or disorder in a subject, for example, cancer, precancer, infection, transplant rejection, or other disorder that causes changes in cell death. In some embodiments, the methods described herein further comprise detecting genetic variants, for example, in the set of sequence variable target regions. In some embodiments, the methods described herein further comprise detecting epigenetic features, for example, DNA methylation and / or fragmentation. In some embodiments, the methods described herein further comprise detecting genetic variants, for example, in the set of sequence variable target regions, and detecting epigenetic features, for example, genomic methylation and / or fragmentation. The detection of epigenetic features can be performed on the set of epigenetic target regions. Exemplary sets of sequence variable target regions and sets of epigenetic target regions are described, for example, in WO2020 / 160414, published August 6, 2020, which is incorporated herein by reference. Detection of genetic variants and / or epigenetic features may be performed using nucleic acid (e.g., cfDNA) from the same sample used to determine the level of the target molecule. Exemplary techniques for detecting genetic variants and / or epigenetic features are described elsewhere herein, including in Section II.D below. In some embodiments, detection of the target molecule in combination with cfDNA analysis of sequence-independent changes in epigenetic target regions, e.g., cfDNA analysis as described herein, indicates the presence of a disease or disorder in the subject, e.g., cancer, precancer, infectious disease, transplant rejection, or other disorder that causes changes in the relative amounts of target molecules associated with cell debris and / or exosomes, as well as DNA changes, compared to healthy subjects. B. Target

[0140] In some embodiments, the sample is obtained from a subject having cancer or precancer, an infection, transplant rejection, or other disease that directly or indirectly affects the immune system. In some embodiments, the sample is obtained from a subject suspected of having cancer or precancer, an infection, transplant rejection, or other disease that directly or indirectly affects the immune system. In some embodiments, the sample is obtained from a subject having a tumor. In some embodiments, the sample is obtained from a subject suspected of having a tumor. In some embodiments, the sample is obtained from a subject having a neoplasm. In some embodiments, the sample is obtained from a subject suspected of having a neoplasm. In some embodiments, the sample is obtained from a subject in remission of the tumor, cancer, or neoplasm (e.g., after chemotherapy, surgical resection, radiation therapy, or a combination thereof). In any of the above-mentioned embodiments, the cancer, tumor, or neoplasm, or suspected cancer, tumor, or neoplasm, may be of the lung, colon, rectum, kidney, breast, prostate, or liver. In some embodiments, the cancer, tumor, or neoplasm, or suspected cancer, tumor, or neoplasm, is of the lung. In some embodiments, the cancer, tumor, or neoplasm, or suspected cancer, tumor, or neoplasm, is of the colon or rectum. In some embodiments, the cancer, tumor, or neoplasm, or suspected cancer, tumor, or neoplasm, is of the breast. In some embodiments, the cancer, tumor, or neoplasm, or suspected cancer, tumor, or neoplasm, is of the prostate. In any of the above embodiments, the subject may be a human subject. C. Analysis

[0141] The method can be used to diagnose the presence of a condition, particularly cancer or precancer, in a subject, characterize the condition (e.g., stage the cancer or determine the heterogeneity of the cancer), monitor the response to the treatment of the condition, and provide a prognostic risk of the development of the condition or the subsequent course of the condition. The present disclosure can also be useful for determining the effectiveness of a particular treatment option. A successful treatment may increase the amount of copy number mutations, rare mutations, or target molecules detected in the subject's blood, since more cancers may die and expel DNA and cellular debris upon successful treatment. In other instances, this may not occur. In another example, perhaps a particular treatment option may correlate with the genetic profile of the cancer over time. This correlation may be useful in selecting a therapy.

[0142] Additionally, if the cancer is observed to be in remission following treatment, the method can be used to monitor for residual disease or recurrence of the disease.

[0143] The types and number of cancers that can be detected include blood cancer, brain cancer, lung cancer, skin cancer, nose cancer, throat cancer, liver cancer, bone cancer, lymphoma, pancreatic cancer, skin cancer, intestinal cancer, rectal cancer, thyroid cancer, bladder cancer, kidney cancer, oral cancer, stomach cancer, solid tumors, heterogeneous tumors, and homogeneous tumors, etc. The type and / or stage of cancer can be detected from gene mutations including mutations, rare mutations, indels, copy number variations, transversions, translocations, recombinations, inversions, deletions, aneuploidy, partial aneuploidy, polyploidy, chromosomal instability, chromosomal structural changes, gene fusions, chromosomal fusions, gene truncations, gene amplifications, gene duplications, chromosomal damage, DNA damage, abnormal changes in nucleic acid chemical modifications, abnormal changes in epigenetic patterns, and abnormal changes in nucleic acid 5-methylcytosine.

[0144] In some embodiments, the methods described herein include identifying the presence of a target molecule and / or DNA produced by a tumor (or neoplastic cell, or cancer cell) or by a precancerous cell. In some embodiments, the methods described herein include determining the level of a target molecule and / or identifying the presence of DNA produced by a tumor (or neoplastic cell, or cancer cell) or by a precancerous cell. In some embodiments, determining the level of the target molecule includes determining either an increased level or a decreased level of the target molecule, where an increased or decreased level of the target molecule is determined by comparing the level of the target molecule to a threshold level / value.

[0145] Genetic data can also be used to characterize specific forms of cancer. Cancers are often heterogeneous in both composition and stage. Genetic profile data can allow characterization of specific subtypes of cancer, which can be important in diagnosing or treating that specific subtype. This information can also provide clues to the subject or attending physician regarding the prognosis of a particular type of cancer, allowing the subject or attending physician to adapt treatment options according to disease progression. Some cancers can progress to become more aggressive and genetically unstable. Other cancers can remain benign, inactive, or dormant. The disclosed system and method can be useful in determining disease progression.

[0146] Furthermore, the method of the present disclosure can be used to characterize the heterogeneity of abnormal conditions in a subject. Such a method may include, for example, generating a profile of extracellular molecules from a subject, the profile including a plurality of data resulting from target molecule detection, and copy number variation, and rare mutation analysis. In some embodiments, the abnormal condition is cancer or precancer. In some embodiments, the abnormal condition may result in a heterogeneous genomic population. In the example of cancer, it is known that some tumors contain tumor cells of different stages of cancer. In other examples, the heterogeneity may include multiple disease foci. Again, in the example of cancer, there may be multiple tumor foci, and perhaps one or more foci are the result of metastasis spreading from the primary site.

[0147] The methods can be used to generate a profile, fingerprint, or dataset that is the sum of information derived from different cells of a heterogeneous disease, which may include the identity and levels of target molecules, copy number variations, epigenetic variations, or other mutational analyses, alone or in combination.

[0148] This method can be used to diagnose, prognose, monitor or observe cancer or other disease.In some embodiments, the method herein does not include diagnosing, prognosing or monitoring fetus, and therefore does not relate to non-invasive prenatal testing.In other embodiments, such methodology can be used in pregnant subjects to diagnose, prognose, monitor or observe cancer or other disease in fetal subjects where DNA and other polynucleotides may co-circulate with maternal molecules. D. DNA analysis; dividing the sample into multiple aliquots

[0149] In some embodiments described herein, the method of the present disclosure further comprises analyzing DNA in a sample (which may be a separate sample from the same subject or may be the same sample). For example, analyzing DNA, such as cell-free DNA, in combination with analysis of target molecules associated with cellular debris can improve the specificity and / or sensitivity of the method to detect an abnormal condition, such as the presence of a disease. As shown in Figures 1A-1B, DNA, such as cell-free DNA, can be isolated from a blood sample or a subsample thereof, such as the plasma supernatant obtained after centrifugation of the plasma. The analysis of DNA may include detection or quantification of the DNA of interest. The analysis of DNA may include detecting genetic variants and / or epigenetic features (e.g., DNA methylation and / or DNA fragmentation).

[0150] In any of these embodiments, the methylation level can be determined using partitioning, methylation-sensitive conversion such as bisulfite conversion, direct detection during sequencing, methylation-sensitive restriction enzyme digestion, or any other suitable technique. For example, different forms of DNA (e.g., hypermethylated DNA and hypomethylated DNA) can be physically partitioned based on one or more characteristics of the DNA. For example, DNA can be partitioned using a methylated DNA binding protein (e.g., MBD such as MBD2, MBD4, or MeCP2) or an antibody specific for 5-methylcytosine (e.g., MeDIP). This technique can be used, for example, to determine whether a particular sequence is hypermethylated or hypomethylated.

[0151] Detecting abnormal DNA features (sequence-based, epigenetic, or both) while also detecting abnormal levels of one or more target molecules in the cellular debris and / or exosomes can provide greater specificity and / or sensitivity for identifying an abnormal condition than detecting a DNA feature alone, or the levels of one or more target molecules in the cellular debris and / or exosomes alone.

[0152] Methylation profiling may include determining methylation patterns across different regions of a genome. For example, after distributing molecules based on the degree of methylation (e.g., the relative number of methylated nucleobases per molecule) and sequencing, the sequences of molecules in different distribution fractions can be mapped against a reference genome. This can show regions of the genome that are more highly methylated or less highly methylated compared to other regions. In this way, genomic regions can have different degrees of methylation as opposed to individual molecules.

[0153] By partitioning the nucleic acid molecules in a sample, rare signals can be increased, for example, by enriching rare nucleic acid molecules that are more abundant in one partitioned fraction of the sample. For example, genetic mutations that are present in hypermethylated DNA but less abundant (or absent) in hypomethylated DNA can be more easily detected by partitioning the sample into hypermethylated and hypomethylated nucleic acid molecules. By analyzing multiple partitioned fractions of a sample, multidimensional analysis of single molecules can be performed, thus achieving higher sensitivity. Partitioning can include physically partitioning nucleic acid molecules into partitioned fractions or subsamples based on the presence or absence of one or more methylated nucleic acid bases. Samples can be partitioned into partitioned fractions or subsamples based on a characteristic that indicates differential gene expression or disease state. Samples can be partitioned based on a characteristic or combination that results in a difference in signal between normal and disease states during the analysis of nucleic acids, for example, cell-free DNA (cfDNA), non-cfDNA, tumor DNA, circulating tumor DNA (ctDNA), and cell-free nucleic acid (cfNA).

[0154] In some embodiments, hypermethylated and / or hypomethylated variable epigenetic target regions are analyzed to determine whether they exhibit differential methylation signatures in tumor cells, or cell types that do not normally contribute to the DNA sample being analyzed (such as cfDNA), and / or specific immune cell types.

[0155] In some cases, the heterogeneous DNA in the sample is distributed into two or more distribution fractions (e.g., at least 3, 4, 5, 6, or 7 distribution fractions). In some embodiments, each distribution fraction is differentially tagged. The tagged distribution fractions can then be pooled together for collective sample preparation and / or sequencing. The distribution-tagging-pooling step may be performed more than once, with each round of distribution being tagged using a differential tag based on a different characteristic (examples are provided herein) and distinguishing it from other distribution fractions and distribution means. In other examples, the differentially tagged distribution fractions are sequenced separately.

[0156] In some embodiments, sequence reads from the differentially tagged pool DNA are obtained and analyzed in silico. The tags are used to sort the reads from different distribution fractions. Analysis to detect genetic mutations can be performed at the level of each distribution fraction as well as the level of the total nucleic acid population. For example, the analysis can include in silico analysis to determine genetic variants such as CNVs, SNVs, indels, fusions, etc. in the nucleic acids of each distribution fraction. In some cases, the in silico analysis can include determining chromatin structure. For example, the coverage of sequence reads can be used to determine nucleosome positions in chromatin. Higher coverage can be correlated with higher nucleosome occupancy in genomic regions, and lower coverage can be correlated with lower nucleosome occupancy or nucleosome depleted regions (NDRs).

[0157] Examples of characteristics that can be used for partitioning include sequence length, methylation level, nucleosome binding, sequence mismatch, immunoprecipitation, and / or proteins that bind to DNA. The resulting partitioned fractions include one or more of the following nucleic acid forms: single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), short DNA fragments, and long DNA fragments. In some embodiments, partitioning is generally performed based on cytosine modification (e.g., cytosine methylation) or methylation, and optionally combined with at least one additional partitioning step that may be based on any of the above-mentioned characteristics or forms of DNA. In some embodiments, a heterogeneous population of nucleic acids is partitioned into nucleic acids that have one or more epigenetic modifications and nucleic acids that do not have one or more epigenetic modifications. Examples of epigenetic modifications include the presence or absence of methylation, the level of methylation, the type of methylation (e.g., 5-methylcytosine versus other types of methylation such as adenine methylation and / or cytosine hydroxymethylation), and the association and level of association with one or more proteins, such as histones. Alternatively or in addition, the heterogeneous population of nucleic acids can be divided into nucleic acid molecules that are associated with nucleosomes and nucleic acid molecules that lack nucleosomes.Alternatively or in addition, the heterogeneous population of nucleic acids can be divided into single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA).Alternatively or in addition, the heterogeneous population of nucleic acids can be divided based on nucleic acid length (e.g., molecules that are up to 160bp and molecules that have a length greater than 160bp).

[0158] The agents used to partition the population of nucleic acids in a sample may be affinity agents such as antibodies, natural binding partners, or variants thereof with the desired specificity (Bock et al., Nat Biotech 28: 1106-1114 (2010); Song et al., Nat Biotech 29: 68-72 (2011)), or artificial peptides selected, for example, by phage display, to have specificity for a given target. In some embodiments, the agents used for partitioning are agents that recognize modified nucleobases. In some embodiments, the modified nucleobase recognized by the agent is a modified cytosine, such as methylcytosine (e.g., 5-methylcytosine). In some embodiments, the modified nucleobase recognized by the agent is the product of a procedure that affects a first nucleobase in DNA differently than a second nucleobase in the DNA of the sample. In some embodiments, the modified nucleobase may be a "converted nucleobase," meaning that its base-pairing specificity has been altered by the procedure. For example, in certain procedures, unmethylated or unmodified cytosine is converted to dihydrouracil, or more generally, at least one modified or unmodified form of cytosine undergoes deamination, resulting in uracil (considered a modified nucleobase in the context of DNA) or a further modified form of uracil. Examples of partitioning agents include antibodies, such as antibodies that recognize modified nucleobases that may be modified cytosines, such as methylcytosine (e.g., 5-methylcytosine). In some embodiments, the partitioning agent is an antibody that recognizes modified cytosines other than 5-methylcytosine, such as 5-carboxylcytosine (5caC). Alternative partitioning agents include the methyl-binding domains (MBDs) and methyl-binding proteins (MBPs) described herein, including proteins such as MeCP2.

[0159] Additional non-limiting examples of partitioning agents are histone-binding proteins that can separate histone-bound nucleic acids from free or unbound nucleic acids. Examples of histone-binding proteins that can be used in the methods disclosed herein include RBBP4, RbAp48, and SANT domain peptides.

[0160] In some embodiments, partitioning may include both binary partitioning and partitioning based on degree / level of modification. For example, methylated fragments may be partitioned by methylated DNA immunoprecipitation (MeDIP), or all methylated fragments may be partitioned from non-methylated fragments using a methyl-binding domain protein (e.g., MethylMinder Methylated DNA Enrichment Kit (ThermoFisher Scientific)). Additional partitioning may then include eluting fragments with different levels of methylation by adjusting the salt concentration in the solution with the methyl-binding domain and bound fragments. With increasing salt concentration, fragments with higher methylation levels are eluted.

[0161] In some cases, the final partitioned fraction is enriched for nucleic acids with different degrees of modification (over- or under-representation of the modification). Over- and under-representation can be defined by comparing the number of modifications made to the nucleic acid with the median number of modifications per strand in the population. For example, if the median number of 5-methylcytosine residues of nucleic acids in a sample is two, then nucleic acids containing more than two 5-methylcytosine residues will have this modification over-represented, and nucleic acids with one or zero 5-methylcytosine residues will have this modification under-represented. The effect of the affinity separation is that the bound phase is enriched for nucleic acids with over-represented modifications, and the non-bound phase (i.e., in solution) is enriched for nucleic acids with under-represented modifications. Nucleic acids in the bound phase can be eluted before further processing.

[0162] Using MeDIP or MethylMiner® Methylated DNA Enrichment Kit (ThermoFisher Scientific), sequential elution can be used to partition different levels of methylation. For example, a low methylation partition fraction (no methylation) can be separated from a methylation partition fraction by contacting the nucleic acid population with the MBD of the kit attached to magnetic beads. The beads are used to separate the methylated nucleic acids from the unmethylated nucleic acids. One or more elution steps are then performed sequentially to elute the nucleic acids with different levels of methylation. For example, a first set of methylated nucleic acids may be eluted with a salt concentration of 160 mM or higher, e.g., at least 150 mM, at least 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1000 mM, or 2000 mM. After such methylated nucleic acids are eluted, magnetic separation is once again used to separate the more highly methylated nucleic acids from the less methylated nucleic acids. The elution and magnetic separation steps can be repeated to generate various distribution fractions, such as a low methylation distribution fraction (enriched for nucleic acids with no methylation), a methylation distribution fraction (enriched for nucleic acids with low levels of methylation), and a high methylation distribution fraction (enriched for nucleic acids with high levels of methylation).

[0163] In some methods, nucleic acids bound to agents used for affinity separation-based partitioning are subjected to a wash step, which washes away nucleic acids that are weakly bound to the affinity agent, and such nucleic acids can be enriched for nucleic acids that have a modification level close to the average or median value (i.e., intermediate between those that remain bound to the solid phase and those that do not upon initial contact of the sample with the agent).

[0164] Affinity separation results in at least two, and sometimes three or more, partitioned fractions of nucleic acids with different degrees of modification. Although the partitioned fractions are still separate, the nucleic acids of at least one partitioned fraction, usually two or three (or more), are linked to a nucleic acid tag, usually provided as a component of an adaptor, such that the nucleic acids of the different partitioned fractions receive different tags that distinguish the members of one partitioned fraction from the members of another partitioned fraction. The tags linked to the nucleic acid molecules of the same partitioned fraction may be the same or different from each other. However, when different from each other, the tags may share part of their code in order to identify the molecules to which they are attached as being from a particular partitioned fraction.

[0165] For further details regarding partitioning nucleic acid samples based on characteristics such as methylation, see WO2018 / 119452, which is incorporated herein by reference.

[0166] In some embodiments, partitioning is performed by contacting the nucleic acid with the methyl-binding domain ("MBD") of a methyl-binding protein ("MBP"). In some such embodiments, the nucleic acid is contacted with the entire MBP. In some embodiments, the MBD binds 5-methylcytosine (5mC), and the MBP comprises an MBD, which is referred to herein interchangeably as a methyl-binding protein or a methyl-binding domain protein. In some embodiments, the MBD is coupled to paramagnetic beads, such as Dynabeads® M-280 streptavidin, via a biotin linker. Partitioning into fractions with different degrees of methylation can be performed by eluting the fractions with increasing NaCl concentrations.

[0167] In some embodiments, the bound DNA is eluted by contacting the antibody or MBD with a protease, such as proteinase K. This may be performed instead of or in addition to the elution step using NaCl as discussed above.

[0168] Examples of modified nucleobase recognizing agents contemplated herein include, but are not limited to, the following: (a) MeCP2 is a protein that preferentially binds 5-methyl-cytosine over unmodified cytosine. (b) RPL26, PRP8, and the DNA mismatch repair protein MHS6 preferentially bind 5-hydroxymethyl-cytosine over unmodified cytosine. (c) FOXK1, FOXK2, FOXP1, FOXP4, and FOXI3 bind preferentially to 5-formyl-cytosine over unmodified cytosine (Iurlaro et al., Genome Biol. 14: R119 (2013)). (d) An antibody specific for one or more methylated or modified nucleobases or their conversion products, such as 5mC, 5caC, or DHU.

[0169] In general, elution is a function of the number of modifications, such as the number of methylation sites per molecule, with increasing salt concentration eluting molecules with more methylation. To elute DNA into different populations based on the degree of methylation, a series of elution buffers with increasing NaCl concentrations can be used. The salt concentration can range from about 100 nM to about 2500 mM NaCl. In one embodiment, the process results in three (3) partitioned fractions. The molecules are contacted with a solution at a first salt concentration that includes molecules that include an agent that recognizes modified nucleobases, and the molecules may be attached to a capture moiety such as streptavidin. At the first salt concentration, some populations of molecules will bind to the agent and some will remain unbound. The unbound population can be separated as a "hypomethylated" population. For example, the first partitioned fraction enriched in hypomethylated forms of DNA is the partitioned fraction that remains unbound at a lower salt concentration, e.g., 100 mM or 160 mM. A second partition fraction enriched for intermediately methylated DNA is eluted using an intermediate salt concentration, for example, between 100 mM and 2000 mM, which is also separated from the sample. A third partition fraction enriched for highly methylated forms of DNA is eluted using a high salt concentration, for example, at least about 2000 mM.

[0170] In some embodiments, methylated DNA is purified using a monoclonal antibody raised against 5-methylcytidine (5mC). To obtain single-stranded DNA fragments, DNA is denatured, for example at 95°C. Protein G coupled to standard or magnetic beads and incubation with anti-5mC antibody followed by washing is used to immunoprecipitate the DNA bound to the antibody. Such DNA can then be eluted. The partitioned fraction may include non-precipitated DNA and one or more partitioned fractions eluted from the beads.

[0171] In some embodiments, the distributed fractions of DNA are desalted and concentrated in preparation for the enzymatic steps of library preparation.

[0172] In some embodiments, methylation is detected using methylation-sensitive conversion. Examples of such conversion techniques include bisulfite conversion, which converts unmodified cytosine and certain modified cytosines (e.g., 5-formylcytosine (fC) or 5-carboxylcytosine (caC)) to uracil, but does not convert other modified cytosines (e.g., 5-methylcytosine, 5-hydroxymethylcytosine). Performing bisulfite conversion can facilitate identifying positions containing mC or hmC using sequence reads. For an exemplary description of bisulfite conversion, see, for example, Moss et al., Nat Commun. 2018; 9: 5068.

[0173] Examples of such conversion techniques include oxidative bisulfite (Ox-BS) conversion. Performing Ox-BS conversion can facilitate identifying the position containing mC using sequence reads. For an exemplary description of oxidative bisulfite conversion, see, for example, Booth et al., Science 2012; 336: 934-937.

[0174] Examples of such conversion techniques also include Tet-assisted bisulfite (TAB) conversion. For example, as described in Yu et al., Cell 2012; 149: 1368-80, β-glucosyltransferase can be used to protect hmC (forming 5-glucosylhydroxymethylcytosine (ghmC)), then mC can be converted to caC using a TET protein such as mTet1, and then bisulfite treatment can be used to convert C and caC to U, while ghmC remains unaffected. Thus, when using TAB conversion, the first nucleobase comprises one or more of unmodified cytosine, fC, caC, mC, or other cytosine forms affected by bisulfite, and the second nucleobase comprises hmC. Performing TAB conversion can facilitate identifying positions that contain hmC using sequence reads.

[0175] Examples of such conversion techniques also include Tet-assisted conversions using substituted borane reducing agents, where appropriate, the substituted borane reducing agent is 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane. See, for example, Liu et al., Nature Biotechnology 2019; 37:424-429 (e.g., attached Figure 1 and attached Note 7). Performing a TAP conversion can facilitate identifying positions containing unmodified C using sequence reads. This procedure encompasses Tet-assisted pyridine borane sequencing (TAPS), which is described in more detail in Liu et al. 2019, supra.

[0176] Alternatively, protection of hmC (e.g., using βGT) may be combined with Tet-assisted conversion with a substituted borane reducing agent. Such implementation of TAPSβ conversion can facilitate using sequence reads to distinguish positions that contain unmodified C or hmC from positions that contain mC on the other hand. For an exemplary description of this type of conversion, see, for example, Liu et al., Nature Biotechnology 2019; 37:424-429.

[0177] Examples of such conversion techniques also include APOBEC coupling epigenetic (ACE) conversion.Performing ACE conversion can facilitate using sequence reads to distinguish between positions that contain hmC and positions that contain mC or unmodified C.For an exemplary description of ACE conversion, see, for example, Schutsky et al., Nature Biotechnology 2018; 36: 1083-1090.

[0178] Examples of such conversion techniques include enzymatic conversion of nucleobases, as in, for example, EM-Seq. See, e.g., Vaisvila R, et al. (2019) EM-seq: Detection of DNA methylation at single base resolution from picograms of DNA. bioRxiv; DOI: 10.1101 / 2019.12.20.884692v1, available at: www.biorxiv.org / content / 10.1101 / 2019.12.20.884692v1.

[0179] In some embodiments, methylation is detected using methylation-sensitive restriction enzymes (MSREs). For example, a portion of a sample can be subjected to digestion with one or more MSREs to cut unmethylated sequences. Exemplary MSREs include AatII, AccII, AciI, Aor13HI, Aor15HI, BspT104I, BssHII, BstUI, Cfr10I, ClaI, CpoI, Eco52I, HaeII, HapII, HhaI, Hin6I, HpaII, HpyCH4IV, MluI, MspI, NaeI, NotI, NruI, NsbI, PmaCI, Psp1406I, PvuI, SacII, SalI, SmaI, and SnaBI. In some embodiments, at least two methylation-sensitive nucleases are used. In some embodiments, at least three methylation-sensitive nucleases are used. In some embodiments, the methylation sensitive nucleases include BstUI and HpaII. In some embodiments, the two methylation sensitive nucleases include HhaI and AccII. In some embodiments, the methylation sensitive nucleases include BstUI, HpaII, and Hin6I. In some embodiments, the portion of the sample contacted with one or more MSREs includes hypermethylated DNA or is or includes a hypermethylated DNA distribution fraction that can be obtained as described elsewhere herein.

[0180] In some embodiments, DNA fragmentation is detected by determining the end point and / or midpoint of the fragment of sequenced DNA (e.g., cfDNA).For example, fragmentation pattern may be different depending on whether fragment originates from tumor or healthy cell. E. Adapter ligation or addition; tagging

[0181] In some embodiments, the disclosed method further comprises analyzing DNA in the samples (which may be separate samples from the same subject or may be the same sample). In such methods, adapters may be added to the DNA. This may be done simultaneously with the amplification procedure, for example, by providing an adapter at the 5' portion of the primer (when PCR is used, this may be called library preparation PCR or LP-PCR). In some embodiments, the adapters are added by other techniques, such as ligation. In some such methods, a first adapter is added to the nucleic acid by ligation to its 3' end, which may include ligation to single-stranded DNA, before partitioning or capture. The adapter can be used as a priming site for second strand synthesis, for example, using a universal primer and a DNA polymerase. A second adapter can then be ligated to at least the 3' end of the second strand of the now double-stranded molecule. In some embodiments, the first adapter comprises an affinity tag, such as biotin, and the nucleic acid ligated to the first adapter is bound to a solid support (e.g., beads), which may comprise a binding partner for the affinity tag, such as streptavidin. For further discussion of related procedures, see Gansauge et al., Nature Protocols 8:737-748 (2013). Commercially available kits for sequencing library preparation that are compatible with single-stranded nucleic acids are available, such as the Accel-NGS® Methyl-Seq DNA Library Kit from Swift Biosciences. In some embodiments, after adaptor ligation, the nucleic acid is amplified.

[0182] Preferably, the adaptors contain a sufficient number of different tags such that the number of tag combinations results in a low probability, e.g., a 95, 99 or 99.9% probability, that two nucleic acids with the same start and stop points will receive the same tag combination. Adaptors, whether they have the same or different tags, may contain the same or different primer binding sites, although preferably the adaptors contain the same primer binding sites.

[0183] In some embodiments, after attachment of the adaptors, the nucleic acid is subjected to amplification, which can, for example, use a universal primer that recognizes the primer binding site of the adaptor.

[0184] In some embodiments, the method includes partitioning the DNA after the attachment of the adaptor and contacting the DNA with an agent that preferentially binds to nucleic acids with epigenetic modifications. The nucleic acid is partitioned into at least two subsamples that differ in the extent to which the nucleic acid has the modification by binding with the agent. For example, if the agent has an affinity for nucleic acids with the modification, nucleic acids with an overabundance of the modification (relative to the median abundance in the population) will preferentially bind to the agent, while nucleic acids with an underabundance of the modification will not bind or will be more easily eluted from the agent. The nucleic acid can then be amplified from a primer that binds to the primer binding site in the adaptor. Alternatively, partitioning may be performed before the attachment of the adaptor, in which case the adaptor may include a differential tag that includes a component that identifies which partition fraction the molecule was in.

[0185] In some embodiments, the nucleic acid is ligated at both ends to a Y-shaped adaptor that comprises a primer binding site and a tag. The molecule is amplified.

[0186] Tagging of DNA molecules is the procedure of attaching or associating a tag to a DNA molecule. Such a tag may be a molecule, such as a nucleic acid, that contains information indicative of a characteristic of the molecule with which the tag is associated. For example, the molecule may have a sample tag (that distinguishes a molecule in one sample from a molecule in another sample), or a molecular tag / molecular barcode / barcode (that distinguishes different molecules from each other (in both unique and non-unique tagging scenarios). In the case of methods that include a partitioning step, it may also include a partitioning tag (that distinguishes a molecule in one partitioned fraction from a molecule in another partitioned fraction). In some embodiments, the adapter that is added to the DNA molecule includes a tag. In certain embodiments, the tag may include a barcode or a combination of barcodes. As used herein, the term "barcode" refers to a nucleic acid molecule having a specific nucleotide sequence, or to the nucleotide sequence itself, depending on the context. A barcode may have, for example, 10-100 nucleotides. A collection of barcodes may have degenerate sequences or sequences with a certain Hamming distance depending on a particular purpose. Thus, for example, a molecular barcode may consist of one barcode or a combination of two barcodes, each attached to a different end of the molecule. Additionally or alternatively, for different distribution fractions and / or samples, different sets of molecular barcodes or molecular tags may be used, such that the barcodes serve as molecular tags due to their individual sequences, and also serve to identify the corresponding distribution fraction and / or sample based on the set of which the barcode is a member.

[0187] In some embodiments, two or more distribution fractions, for example each distribution fraction, are differentially tagged. Tags can be used to label individual polynucleotide population distribution fractions, such that the tag (or tags) are correlated with a particular distribution fraction. Alternatively, tags can be used in embodiments that do not use a distribution step. In some embodiments, a single tag can be used to label a particular distribution fraction. In some embodiments, multiple different tags can be used to label a particular distribution fraction. In embodiments where multiple different tags are used to label a particular distribution fraction, the set of tags used to label one distribution fraction can be easily distinguished from the set of tags used to label other distribution fractions. In some embodiments, the tag may have additional functionality, e.g., the tag may be used to index the sample source or may be used as a unique molecular identifier (which may be used to improve the quality of sequencing data by distinguishing sequencing errors from mutations, e.g., as in Kinde et al., Proc Nat'l Acad Sci USA 108: 9530-9535 (2011); Kou et al., PLoS ONE,11: e0146638 (2016)), or may be used as a non-unique molecular identifier, e.g., as described in U.S. Pat. No. 9,598,731. Similarly, in some embodiments, the tag may have additional functionality, e.g., the tag may be used to index the sample source or may be used as a non-unique molecular identifier (which may be used to improve the quality of sequencing data by distinguishing sequencing errors from mutations).

[0188] In some embodiments, the distribution fraction tagging comprises tagging the molecules in each distribution fraction with a distribution tag. After remixing the distribution fractions (e.g., to reduce the number of required sequencing runs and avoid unnecessary costs) and sequencing the molecules, the source distribution fraction is identified by the distribution tag. In another embodiment, different distribution fractions are tagged with different sets of molecular tags, e.g., composed of a pair of barcodes. In this way, each molecular barcode indicates the source distribution fraction, as well as being useful for distinguishing molecules within the distribution fraction. For example, a first set of 35 barcodes can be used to tag the molecules in the first distribution fraction, and a second set of 35 barcodes can be used to tag the molecules in the second distribution fraction.

[0189] In some embodiments, after partitioning and tagging with partitioning tags, molecules may be pooled for sequencing in a single run. In some embodiments, sample tags are added to molecules, for example, in a step following partitioning tag addition and pooling. Sample tags can facilitate pooling of material generated from multiple samples for sequencing in a single sequencing run.

[0190] Alternatively, in some embodiments, the distribution tag can be correlated with the sample and the distribution fraction.As a simple example, the first tag can indicate the first distribution fraction of the first sample, the second tag can indicate the second distribution fraction of the first sample, the third tag can indicate the first distribution fraction of the second sample, and the fourth tag can indicate the second distribution fraction of the second sample.

[0191] Tags may be attached to molecules that have already been distributed based on one or more characteristics, but the final tagged molecules in the library may no longer have those characteristics. For example, single-stranded DNA molecules may be distributed and tagged, but the final tagged molecules in the library are likely to be double-stranded. Similarly, DNA may be subjected to distribution based on different levels of methylation, but the tagged molecules derived from such molecules in the final library may not be methylated. Thus, the tags attached to molecules in the library typically represent the characteristics of the "parent molecule" from which the final tagged molecule is derived, and not necessarily the characteristics of the tagged molecule itself.

[0192] As an example, the molecules in the first distribution fraction are tagged and labeled using barcodes 1, 2, 3, 4, etc. The molecules in the second distribution fraction are tagged and labeled using barcodes A, B, C, D, etc. The molecules in the third distribution fraction are tagged and labeled using barcodes a, b, c, d, etc. The differentially tagged distribution fractions may be pooled before sequencing. The differentially tagged distribution fractions may be sequenced separately or may be sequenced together at the same time, for example, in the same flow cell of an Illumina sequencer.

[0193] After sequencing, analysis of reads can be performed at the level of each distribution fraction as well as at the level of the total DNA population. Tags are used to sort the reads of different distribution fractions. Analysis can include in silico analysis to determine genetic and epigenetic mutations (one or more of methylation, chromatin structure, etc.) using sequence information, genome coordinate length, coverage, and / or copy number. In some embodiments, higher coverage can be correlated with higher nucleosome occupancy in genomic regions, and lower coverage can be correlated with lower nucleosome occupancy or nucleosome depleted regions (NDRs).

[0194] Molecular tagging refers to a tagging operation that allows the DNA molecule from which a sequence read originates to be differentiated. Tagging strategies can be divided into unique and non-unique tagging strategies. In unique tagging, all or substantially all molecules in a sample have different tags, so that the reads can be assigned to the original molecule based on tag information alone. The tags used in such methods are sometimes called "unique tags". In non-unique tagging, different molecules in the same sample may have the same tag, so that other information in addition to the tag information is used to assign the sequence read to the original molecule. Such information can include start and end coordinates, coordinates to which the molecule is mapped, start coordinates alone or end coordinates alone, etc. The tags used in such methods are sometimes called "non-unique tags". Thus, it is not necessary to uniquely tag every molecule in a sample. It is sufficient to uniquely tag molecules that fall into a distinguishable class in a sample. Thus, molecules of different distinguishable families can have the same tag without losing information about the identity of the tagged molecule.

[0195] In certain embodiments of non-unique tagging, the number of different tags used may be sufficient to make it highly likely (e.g., at least 99%, at least 99.9%, at least 99.99%, or at least 99.999%) that all DNA molecules in a particular group have different tags. Note that when barcodes are used as tags, and when barcodes are attached, for example, randomly, to both ends of the molecules, a combination of barcodes together may constitute a tag. And this number is a function of the number of molecules that are classified into that call. For example, a class may be all molecules that map to the same start-end position of the reference genome. A class may be all molecules that map to a particular locus, for example, a particular base or across a particular region (e.g., up to 100 bases or a gene or exon of a gene). In certain embodiments, the number of different tags used to uniquely identify a number z of molecules in a class can be from 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., a lower limit) to any of 100,000*z, 10,000*z, 1000*z, or 100*z (e.g., an upper limit).

[0196] For example, in a sample of about 5 ng-30 ng of cell-free DNA, roughly 3000 molecules are expected to map to a particular nucleotide coordinate, with about 3-10 molecules with any given start coordinate expected to share the same end coordinate. Thus, about 50 to about 50,000 different tags (e.g., about 6-220 barcode combinations) may be sufficient to uniquely tag all such molecules. About 1 million to about 20 million different tags would be required to uniquely tag all 3,000 molecules that map across nucleotide coordinates.

[0197] In general, assignment of unique or non-unique tag barcodes in the reactions follows the methods and systems described in U.S. Patent Application Publication Nos. 20010053519, 20030152490, 20110160078, and U.S. Patent Nos. 6,582,908, 7,537,898, and 9,598,731. Tags may be randomly or non-randomly linked to the sample nucleic acids.

[0198] In some embodiments, the tagged nucleic acid is sequenced after loading into microwell plate.Microwell plate may have 96,384 or 1536 microwells.In some cases, they are introduced into microwell with expected ratio of unique tags.For example, unique tags can be loaded so that more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 50,000,000, or 1,000,000,000 unique tags are loaded per genome sample. In some cases, unique tags can be loaded such that less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 50,000,000, or 1,000,000,000 unique tags are loaded per genomic sample. In some cases, the average number of unique tags loaded per sample genome is less than or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 50,000,000, or 1,000,000,000 unique tags per genome sample.

[0199] In a preferred format, 20-50 different tags (e.g., barcodes) ligated to both ends of a target nucleic acid are used. For example, 35 different tags (e.g., barcodes) are ligated to both ends of a target molecule to create 35x35 permutations, which is equivalent to 1225 for 35 tags. Such a number of tags is sufficient to ensure a high probability (e.g., at least 94%, 99.5%, 99.99%, 99.999%) that different molecules with the same start and end points will receive different combinations of tags. Other barcode combinations include any number between 10 and 500, such as about 15x15, about 35x35, about 75x75, about 100x100, about 250x250, about 500x500.

[0200] In some cases, the unique tag may be an oligonucleotide of predetermined or random or semi-random sequence. In other cases, multiple barcodes may be used, and the multiple barcodes are not necessarily unique to each other. In this example, the barcode can be ligated to each molecule, such that the combination of the barcode and the sequence to which it can be ligated creates a unique sequence that can be tracked individually. As described herein, detection of sequence data non-unique barcodes in combination with the beginning (start) and end (end) parts of sequence reads can allow for the assignment of a unique identity to a particular molecule. The length or number of base pairs of each sequence read can also be used to assign a unique identity to such a molecule. As described herein, fragments derived from a single strand of nucleic acid that are assigned a unique identity can thereby allow subsequent identification of fragments derived from the parent strand. F. Enrichment / capture step; Amplification

[0201] The methods disclosed herein may include enriching, capturing, or isolating complexes, such as complexes containing cell debris and target molecules, and / or enriching, capturing, or isolating DNA, such as cfDNA target regions. In some embodiments, capturing includes contacting the complexes or target molecules with cell debris markers and / or exosome markers or binding molecules specific to the target molecules, and / or contacting DNA with probes specific to the target regions. Enrichment or capture can be performed on any sample or subsample described herein using any suitable technique known in the art.

[0202] In some embodiments, the binding molecule specific for the marker or target molecule, or the probe specific for the DNA target region, comprises a capture moiety that facilitates enrichment or capture of the target molecule, or DNA hybridized to the probe, respectively. In some embodiments, the capture moiety is biotin. In some such embodiments, streptavidin attached to a solid support, such as a magnetic bead, is used to bind to the biotin. In some embodiments, non-specifically bound DNA that does not contain the target region is washed away from the captured DNA. In some embodiments, the DNA is then dissociated from the probe and eluted from the solid support using a buffer containing a salt wash or another DNA denaturing agent. In some embodiments, the probe is also eluted from the solid support, for example, by disrupting the biotin-streptavidin interaction. In some embodiments, the captured DNA is amplified after elution from the solid support. In some such embodiments, the DNA containing the adapter is amplified using a PCR primer that anneals to the adapter. In some embodiments, the captured DNA is amplified while still attached to the solid support. In some such embodiments, amplification involves the use of a PCR primer that anneals to a sequence within the adapter and a PCR primer that anneals to a sequence within the probe that anneals to the target region of DNA.

[0203] In some embodiments, the methods herein include enriching or capturing DNA comprising epigenetic and / or sequence variable target regions. Such regions may be captured from an aliquot of a sample (e.g., a sample that has undergone adapter attachment and amplification), while a step of distributing the DNA with an agent that recognizes methylcytosine is performed on another aliquot of the sample. Enriching or capturing DNA comprising epigenetic and / or sequence variable target regions may include contacting the DNA with a first or second set of target specific probes. Such target specific probes may have any of the characteristics described herein for a set of target specific probes, including but not limited to those in the embodiments shown above and in the section on probes below. Capture may be performed on one or more aliquots prepared by the methods disclosed herein. In some embodiments, DNA is captured from a first aliquot or a second aliquot, e.g., a first aliquot and a second aliquot. In some embodiments, the aliquots are differentially tagged (e.g., as described herein) and then pooled before undergoing capture. Exemplary methods for capturing DNA containing epigenetic and / or sequence variable target regions can be found, for example, in WO2020 / 160414, which is hereby incorporated by reference herein.

[0204] The capture step can be carried out using the conditions suitable for specific nucleic acid hybridization, which generally depend to some extent on the characteristics of the probe, such as length, base composition, etc. Those skilled in the art will be familiar with the appropriate conditions, taking into account the general knowledge in the technical field of nucleic acid hybridization. In some embodiments, a complex of target-specific probe and DNA is formed.

[0205] In some embodiments, the method described herein comprises capturing a plurality of sets of target regions of cfDNA obtained from a subject. The target regions may contain differences depending on whether they originate from a tumor or from a healthy cell or from a certain cell type. In the capture step, a capture set of cfDNA molecules is generated. In some embodiments, cfDNA molecules corresponding to the set of sequence-variable target regions are captured with a higher capture yield in the capture set of cfDNA molecules than cfDNA molecules corresponding to the set of epigenetic target regions. In some embodiments, the method described herein comprises contacting cfDNA obtained from a subject with a set of target-specific probes, the set of target-specific probes being configured to capture cfDNA corresponding to the set of sequence-variable target regions with a higher capture yield than cfDNA corresponding to the set of epigenetic target regions. For additional discussion of the capture step, capture yield, and related aspects, see WO2020 / 160414, which is incorporated herein by reference for all purposes.

[0206] Because analysis of sequence variable target regions may require deeper sequencing depth with sufficient reliability or accuracy than may be required for analysis of epigenetic target regions, it may be beneficial to capture cfDNA corresponding to sequence variable target region sets with higher capture yield than cfDNA corresponding to epigenetic target region sets. The amount of data required to determine fragmentation patterns (e.g., to investigate breakage of transcription start sites or CTCF binding sites) or fragment abundance (e.g., in high-methylation and low-methylation distribution fractions) is generally less than the amount of data required to determine the presence or absence of cancer-associated sequence mutations. Capturing target region sets with different yields may facilitate sequencing target regions to different sequencing depths in the same sequencing run (e.g., using pooled mixtures and / or in the same sequencing cell).

[0207] In some embodiments, DNA is amplified. In some embodiments, amplification is performed before the capture step. In some embodiments, amplification is performed after the capture step. In some embodiments, amplification is performed before and after the capture step. In various embodiments, the method further comprises sequencing the captured DNA, for example, to different degrees of sequencing depth for the epigenetic target region set and the sequence variable target region set, as consistent with the discussion herein.

[0208] In some embodiments, the capture step is performed simultaneously in the same vessel using probes for a set of sequence variable target regions and probes for a set of epigenetic target regions, e.g., the probes for the set of sequence variable target regions and the set of epigenetic target regions are of the same composition. This approach provides a relatively simple workflow.

[0209] In some embodiments, the adapter is included in the DNA, as described herein. In some embodiments, the tag may be or include a barcode and is included in the DNA. In some embodiments, such a tag is included in the adapter. The tag can facilitate identification of the origin of the nucleic acid. For example, the barcode can allow identification of the source (e.g., subject) from which the DNA originated after pooling multiple samples for parallel sequencing. This can be done simultaneously with the amplification procedure, for example by providing a barcode in the 5' portion of the primer, as described herein. In some embodiments, the adapter and the tag / barcode are provided by the same primer or primer set. For example, the barcode may be located 3' of the adapter and 5' of the target hybridization portion of the primer. Alternatively, the barcode can be added by other techniques, such as ligation, if desired, with the adapter of the same ligation substrate.

[0210] Additional details regarding amplification, tags, and barcodes are discussed herein, which may be combined to the extent practicable with any of these embodiments. G. Capture set; target region

[0211] In some embodiments, the nucleic acid captured or enriched using the methods described herein comprises capture DNA, such as one or more capture sets of DNA.In some embodiments, the capture DNA comprises a target region that is differentially methylated in different immune cell types.In some embodiments, the immune cell types comprise rare or closely related immune cell types, such as activated lymphocytes and naive lymphocytes or myeloid cells at different stages of differentiation.

[0212] In some embodiments, the captured epigenetic target region set captured from the sample or the first subsample comprises a hypermethylated variable target region. In some embodiments, the hypermethylated variable target region is differentially or exclusively hypermethylated in one cell type, or in one immune cell type, or in one immune cell type in a cluster. In some embodiments, the hypermethylated variable target region is hypermethylated to a degree that it is significantly more prevalent or exclusively present in one cell type, or in one immune cell type, or in one immune cell type in a cluster. Such a hypermethylated variable target region may be hypermethylated in other cell types, but not to the extent observed in the one cell type. In some embodiments, the hypermethylated variable target region shows lower methylation in healthy cfDNA than at least one other tissue type.

[0213] In some embodiments, the captured epigenetic target region set captured from the sample or the second subsample comprises a hypomethylated variable target region. In some embodiments, the hypomethylated variable target region is exclusively hypomethylated in one cell type, or in one immune cell type, or in one immune cell type in a cluster. In some embodiments, the hypomethylated variable target region is hypomethylated to the extent that it is exclusively present in one cell type, or in one immune cell type, or in one immune cell type in a cluster. Such a hypomethylated variable target region may be hypomethylated in other cell types, but not to the extent observed in the one cell type. In some embodiments, the hypomethylated variable target region shows higher methylation in healthy cfDNA than at least one other tissue type.

[0214] Without wishing to be bound by any particular theory, in individuals with cancer, proliferating or activated immune cells and / or cancer cells may shed more DNA into the bloodstream than immune cells of healthy individuals and / or healthy cells of the same tissue type, respectively. Therefore, the distribution of cell types and / or tissues from which cfDNA originates may change during carcinogenesis. Thus, variations in hypermethylation and / or hypomethylation may be indicative of disease. For example, an increase in the levels of hypermethylated and / or hypomethylated variable target regions in the partial samples after the partitioning step may be indicative of the presence (or recurrence depending on the subject's medical history) of cancer.

[0215] Exemplary hypermethylated and hypomethylated variable target regions useful for distinguishing various cell types, including but not limited to immune cell types, have been identified by analyzing DNA from various cell types by whole genome bisulfite sequencing, as described, for example, in Scott, CA, Duryea, JD, MacKay, H. et al., "Identification of cell type-specific methylation signals in bulk whole genome bisulfite sequencing data," Genome Biol 21, 156 (2020) (doi.org / 10.1186 / s13059-020-02065-5). Whole genome bisulfite sequence data is available from the Blueprint Consortium and available on the internet at dcc.blueprint-epigenome.eu.

[0216] In some embodiments, the first and second capture target region sets comprise DNA corresponding to a sequence variable target region set and DNA corresponding to an epigenetic target region set, respectively, for example as described in WO2020 / 160414. The first and second capture sets may be mixed to provide a mixed capture set. The sequence variable target region set and the epigenetic target region set may have any of the features described for such sets in WO2020 / 160414, the entirety of which is incorporated herein by reference. In some embodiments, the epigenetic target region set comprises a hypermethylated variable target region set. In some embodiments, the epigenetic target region set comprises a hypomethylated variable target region set. In some embodiments, the epigenetic target region set comprises a CTCF binding region. In some embodiments, the epigenetic target region set comprises a fragmented variable target region. In some embodiments, the epigenetic target region set comprises a transcription start site. In some embodiments, the set of epigenetic target regions includes one or more of the regions that may show local amplification in cancer, such as AR, BRAF, CCND1, CCND2, CCNE1, CDK4, CDK6, EGFR, ERBB2, FGFR1, FGFR2, KIT, KRAS, MET, MYC, PDGFRA, PIK3CA, and RAF1. For example, in some embodiments, the set of epigenetic target regions includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the above targets.

[0217] In some embodiments, the sequence variable target region set includes a plurality of regions known to undergo somatic mutations in cancer. In some aspects, the sequence variable target region set targets a plurality of different genes or genomic regions ("panels") selected such that a determined percentage of subjects with cancer exhibits genetic variants or tumor markers in one or more different genes or genomic regions in the panel. The panel can be selected to restrict the region for sequencing to a certain number of base pairs. The panel may be selected to sequence a desired amount of DNA, for example, by adjusting the affinity and / or amount of probes as described elsewhere herein. The panel may further be selected to achieve a desired sequence read depth. The panel may be selected to achieve a desired sequence read depth or sequence read coverage relative to the amount of base pairs sequenced. The panel may be selected to achieve a theoretical sensitivity, theoretical specificity, and / or theoretical accuracy for detecting one or more genetic variants in a sample.

[0218] The probe for detecting a panel of regions can include the probe for detecting genomic regions of interest (hotspot regions).The design of the probe can take into account information about chromatin structure, and / or the probe can be designed to maximize the possibility that a particular site (e.g., KRAS codons 12 and 13) can be captured, and can be designed to optimize capture based on the analysis of cfDNA coverage and fragment size variation that is influenced by nucleosome binding patterns and GC sequence composition.In addition, the region used herein can include non-hotspot regions that are optimized based on nucleosome position and GC model.

[0219] Examples of lists of genomic locations of interest can be found in Tables 3 and 4 of WO2020 / 160414. In some embodiments, the sequence variable target region set used in the method of the present disclosure comprises at least a portion of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or 70 genes in Table 3 of WO2020 / 160414. In some embodiments, the sequence variable target region set used in the method of the present disclosure comprises at least a portion of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, or 73 genes in Table 4 of WO2020 / 160414. Additionally or alternatively, suitable target region sets are available from the literature. For example, Gale et al., PLoS One 13: e0194630 (2018), incorporated herein by reference, describes a panel of 35 cancer-related gene targets that can be used as part or all of a sequence mutation target region set.These 35 targets are AKT1, ALK, BRAF, CCND1, CDK2A, CTNNB1, EGFR, ERBB2, ESR1, FGFR1, FGFR2, FGFR3, FOXL2, GATA3, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, KIT, KRAS, MED12, MET, MYC, NFE2L2, NRAS, PDGFRA, PIK3CA, PPP2R1A, PTEN, RET, STK11, TP53, and U2AF1.

[0220] In some embodiments, the set of sequence variable target regions includes target regions of at least 10, 20, 30, or 35 cancer-associated genes, such as those listed above and in Tables 3 and 4 of WO2020 / 160414. H. Sequencing

[0221] Generally, sample protein and / or nucleic acid, including nucleic acid flanked by adaptor, can be subjected to sequencing with or without prior amplification.Sequencing methods include, for example, Edman degradation-based protein sequencing, mass spectrometry-based protein sequencing, Sanger sequencing, high-throughput sequencing, pyrosequencing, sequencing by synthesis, single molecule sequencing, nanopore sequencing, semiconductor sequencing, ligation sequencing, hybridization sequencing, 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 platform.

[0222] In some embodiments, the sequencing method includes detecting and / or distinguishing between unmodified and modified nucleobases. For example, single molecule real-time (SMRT) sequencing facilitates direct detection of, for example, 5-methylcytosine and 5-hydroxymethylcytosine as well as unmodified cytosine. See, for example, Schatz., Nature Methods. 14(4): 347-348 (2017) and US9,150,918. The sequencing reaction can be carried out in a variety of sample processing units, which may be multiple lanes, multiple channels, multiple wells, or other means of processing multiple sample sets substantially simultaneously. Also, the sample processing unit may include multiple sample chambers so that multiple runs can be processed simultaneously.

[0223] The sequencing reaction can be performed on one or more forms of nucleic acid, such as nucleic acids known to contain markers for cancer or other diseases. The sequencing reaction can also be performed on any nucleic acid fragments present in the sample. In some embodiments, the sequence coverage of the genome can be less than 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or 100%. In some embodiments, the sequencing reaction can provide sequence coverage of at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of the genome. Sequence coverage can be performed for at least 5, 10, 20, 70, 100, 200, or 500 different genes, or up to 5000, 2500, 1000, 500, or 100 different genes.

[0224] Simultaneous sequencing reaction can be carried out using multiplex sequencing.In some cases, cell-free nucleic acid can be sequenced using at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, 100,000 sequencing reactions.In other cases, cell-free nucleic acid can be sequenced using less than 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, 100,000 sequencing reactions.Sequencing reactions can be carried out sequentially or simultaneously.Subsequent data analysis can be carried out on all or part of sequencing reactions. In some cases, data analysis may be performed on at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, 100,000 sequencing reactions. In other cases, data analysis may be performed on less than 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, 100,000 sequencing reactions. Exemplary read depths are 1000-50000 reads per locus (base). III. ADDITIONAL FEATURES OF CERTAIN DISCLOSED METHODS A. Sample

[0225] The sample may be any biological sample isolated from a subject. The sample may be a body sample. The sample may include body tissues or fluids such as known or suspected solid tumors, whole blood, platelets, serum, plasma, stool, red blood cells, white blood cells or leukocytes, endothelial cells, tissue biopsy, cerebrospinal fluid, synovial fluid, lymphatic fluid, ascites, interstitial or extracellular fluid, fluid in the space between cells, gingival crevicular fluid, bone marrow, pleural fluid, pleural fluid, cerebrospinal fluid, saliva, mucus, sputum, semen, sweat, and urine. The sample is preferably a body fluid, in particular blood and its fractions, cerebrospinal fluid, pleural fluid, saliva, sputum, or urine. The sample may be in the original form isolated from the subject, or may have been subjected to further processing to remove or add components such as cells, or to enrich one component relative to another. Thus, the preferred body fluid for analysis is plasma or serum, which contains cell-free nucleic acid.

[0226] In some embodiments, the population of nucleic acids is obtained from serum, plasma or blood samples from subjects suspected of having neoplasm, tumor, precancer or cancer, or from subjects previously diagnosed with neoplasm, tumor, precancer or cancer.The population comprises nucleic acids with various levels of sequence mutation, epigenetic mutation, and / or post-replicative or post-transcriptional modification.Post-replicative modifications include, in particular, modifications of cytosine at the 5th position of nucleic acid base, such as 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine.

[0227] The sample can be isolated or obtained from the subject and transported to the site of sample analysis. The sample can be stored and shipped at a desired temperature, for example, room temperature, 4°C, -20°C, and / or -80°C. The sample can be isolated or obtained from the subject at the site of sample analysis. The subject can be a human, a mammal, an animal, a companion animal, a service animal, or a pet. The subject can have cancer, precancer, an infection, transplant rejection, or other disease or disorder related to alterations in the immune system. The subject can have no cancer or detectable symptoms of cancer. The subject can be treated with one or more cancer therapies, for example, any one or more of chemotherapy, antibodies, vaccines, or biologics. The subject can be in remission. The subject may or may not have been diagnosed with cancer or susceptible to any cancer-related genetic mutation / disorder.

[0228] In some embodiments, the sample comprises plasma. The volume of plasma obtained may depend on the desired read depth of the sequenced region. Exemplary volumes are 0.4-40 ml, 5-20 ml, 10-20 ml. For example, the volume may be 0.5 mL, 1 mL, 5 mL 10 mL, 20 mL, 30 mL, or 40 mL. The volume of plasma collected may be 5-20 mL. B. Capturing part

[0229] As discussed above, molecules such as proteins and / or nucleic acids in a sample may be subjected to a capture step in which target molecules or molecules with target regions are captured and analyzed. Target capture may include the use of an oligonucleotide labeled with a capture moiety, such as biotin, and a second moiety or binding partner that binds to the capture moiety, such as streptavidin. In some embodiments, the capture moiety and the binding partner may have higher and lower capture yields for different sets of target regions, such as the capture yields of a sequence-variable target region set and an epigenetic target region set, respectively, as discussed elsewhere herein. Methods involving capture moieties are further described, for example, in U.S. Patent No. 9,850,523, issued December 26, 2017. This document is incorporated herein by reference.

[0230] Capture moieties include, but are not limited to, biotin, avidin, streptavidin, nucleic acids containing specific nucleotide sequences, haptens recognized by antibodies, and magnetically attractive particles. Extraction moieties may be members of binding pairs, such as biotin / streptavidin or hapten / antibody. In some embodiments, the capture moiety attached to the analyte is captured by its binding partner attached to a separable moiety, such as a magnetically attractive particle or a large particle that can be sedimented by centrifugation. The capture moiety may be any type of molecule that allows affinity separation of nucleic acids that have a capture moiety and nucleic acids that lack a capture moiety. Exemplary capture moieties are biotin, which allows affinity separation by binding to streptavidin that is linked or linkable to a solid phase, or oligonucleotides that allow affinity separation by binding to complementary oligonucleotides that are linked or linkable to a solid phase. C. Computer Systems

[0231] The methods of the present disclosure can be implemented using or with the assistance of a computer system. Figure 2 shows a computer system 201 programmed or otherwise configured to implement the methods of the present disclosure. The computer system 201 can control various aspects of sample preparation, sequencing, and / or analysis. In some examples, the computer system 201 is configured to perform sample analysis, including sample preparation and nucleic acid sequencing (if applicable), for example, according to any of the methods disclosed herein.

[0232] The computer system 201 includes a central processing unit (CPU, also referred to herein as "processor" and "computer processor") 205, which may be a single-core or multi-core processor, or multiple processors for parallel processing. The computer system 201 also includes memory or memory locations 210 (e.g., random access memory, read-only memory, flash memory), electronic storage 215 (e.g., hard disk), communication interface 220 (e.g., network adapter) for communication with one or more other systems, and peripheral devices 225, such as cache, other memory, data storage devices, and / or electronic display adapters. The memory 210, storage 215, interface 220, and peripheral devices 225 communicate with the CPU 205 via a communication network or bus (solid lines), such as a motherboard. The storage 215 may be a data storage device (or data repository) for storing data. The computer system 201 may be operatively connected to a computer network 230 with the aid of the communication interface 220. The computer network 230 may be the Internet, an Internet and / or an extranet, or an intranet and / or an extranet in communication with the Internet. The computer network 230 may in some cases be a telecommunications network and / or a data network. The computer network 230 may include one or more computer servers that may enable distributed computing, such as cloud computing. The computer network 230 may in some cases implement a peer-to-peer network that may enable devices connected to the computer system 201 to act as clients or servers, with the assistance of the computer system 201.

[0233] CPU 205 may execute sequences of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as memory 210. Examples of operations performed by CPU 205 may include fetch, decode, execute, and writeback.

[0234] The storage device 215 can store files such as drivers, libraries, and saved programs. The storage device 215 can store user generated programs and recorded sessions, as well as output(s) associated with the programs. The storage device 215 can store user data, such as user selections and user programs. The computer system 201 may, in some cases, include one or more additional data storage devices that are external to the computer system 201, such as located on a remote server that communicates with the computer system 201 via an intranet or the Internet. Data can be transferred from one location to another, for example, using a communications network or physical data transfer (e.g., using a hard drive, thumb drive, or other data storage mechanism).

[0235] Computer system 201 can communicate with one or more remote computer systems via network 230. In an embodiment, computer system 201 can communicate with a remote computer system of a user (e.g., an operator). Examples of remote computer systems include a personal computer (e.g., a portable PC), a slate or tablet PC (e.g., Apple® iPad®, Samsung® Galaxy Tab), a phone, a smartphone (e.g., Apple® iPhone®, Android®-enabled device, Blackberry®), or a personal digital assistant. A user can access computer system 201 via network 230.

[0236] The methods described herein may be implemented by machine (e.g., a computer processor) executable code stored in an electronic storage location of the computer system 201, such as, for example, memory 210 or electronic storage 215. The machine executable or machine readable code may be provided in the form of software. During use, the processor 205 may execute the code. In some cases, the code may be retrieved from storage 215 and stored in memory 210 for immediate access by the processor 205. In some cases, the electronic storage 215 may be omitted and the machine executable instructions are stored in memory 210.

[0237] In one aspect, the present disclosure provides a non-transitory computer-readable medium comprising computer-executable instructions that, when executed by at least one electronic processor, perform at least a portion of the method described herein.For example, the method may include: collecting a sample from a subject and optionally fractionating the sample; contacting the sample or a subsample thereof with at least one cell debris marker binding molecule; capturing a complex containing the cell debris marker binding molecule or directly detecting a target molecule associated with the complex; detecting and identifying the level of the target molecule, the likelihood that the subject has cancer or other disease, and / or an appropriate treatment for cancer or other disease.

[0238] The code may be pre-compiled and configured for use on a machine having a processor adapted to execute the code, or may be compiled at run-time. The code may be provided in a programming language that may be selected to enable the code to be executed in a pre-compiled or co-compiled manner.

[0239] Aspects of the systems and methods provided herein, such as computer system 201, can be embodied in programming. Various aspects of the technology can be considered to be "products" or "articles" in the form of machine (or processor) executable code and / or associated data, typically carried or embodied in some type of machine-readable medium. The machine-executable code can be stored in electronic storage, such as memory (e.g., read-only memory, random access memory, flash memory) or hard disk. A "storage" type medium can include any or all of the tangible memory, such as a computer or processor, or associated modules thereof, such as various semiconductor memories, tape drives, and disk drives, that can provide non-transitory storage at any time for software programming.

[0240] All or parts of the software may be communicated from time to time via the Internet or various other telecommunications networks. Such communication may, for example, allow the software to be loaded from one computer or processor to another, for example, from a management server or host computer to a computer platform of an application server. Thus, other types of media that may carry software elements include optical, electrical, and electromagnetic waves, such as those used between physical interfaces between local devices over wired and optical landline networks, and through various wireless links. Physical elements that transmit such waves, such as wired or wireless or optical links, may also be considered to be media that carry the software. As used herein, unless limited to non-transitory tangible "storage" media, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions for execution to a processor.

[0241] Thus, a machine-readable medium such as a computer executable code can take many forms, including but not limited to a tangible storage medium, a carrier wave medium, or a physical transmission medium. Non-volatile storage media include optical or magnetic disks, such as any of the storage devices of any computer(s), such as those shown in the drawings, that can be used to implement databases, etc. Volatile storage media include dynamic memories, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that make up a bus in 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. Thus, common forms of computer readable media include, for example, floppy disks, flexible disks, hard disks, magnetic tape, any other magnetic media, CD-ROMs, DVDs or DVD-ROMs, any other optical media, punch cards, paper tape, any other physical storage media having a pattern of holes, RAM, ROM, PROMs and EPROMs, Flash-EPROMs, any other memory chips or cartridges, carrier waves carrying data or instructions, cables or links carrying such carrier waves, or any other medium from which a computer can read programming code and / or data. Many of these forms of computer readable media can be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0242] The computer system 201 can include or communicate with, for example, an electronic display with a user interface (UI) for providing one or more results of a sample analysis. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0243] Additional details regarding computer systems and networks, databases, and computer program products are found in, for example, Peterson, Computer Networks: A Systems Approach, Morgan Kaufmann, 5th Ed. (2011); Kurose, Computer Networking: A Top-Down Approach, Pearson, 7 th Ed. (2016), Elmasri, Fundamentals of Database Systems, Addison Wesley, 6th Ed. (2010), Coronel, Database Systems: Design, Implementation, & Management, Cengage Learning, 11 th Ed. (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 incorporated herein by reference in its entirety. D. Application 1. Cancer and other diseases

[0244] The method can be used to diagnose the presence of a condition, particularly cancer or precancer, in a subject, characterize the condition (e.g., stage the cancer or determine the heterogeneity of the cancer), monitor the response to the treatment of the condition, and achieve a prognostic risk of the onset of the condition or the subsequent course of the condition. The present disclosure can also be useful in determining the effectiveness of a particular treatment option. With successful treatment, more cancers may die and expel cellular debris, so the success of the treatment option may increase the amount of target molecules, copy number mutations, or rare mutations detected in the subject's blood. In other examples, this may not occur. In another example, a particular treatment option may correlate with the cancer profile (e.g., of cellular debris-associated proteins and / or genetic profile) over time. This correlation can be useful in selecting a therapy.

[0245] In some embodiments, the method is used in cancer screening or for cancer screening.For example, the sample may be from a subject who has not been diagnosed with cancer before.In some embodiments, the subject may or may not have cancer.In some embodiments, the subject may or may not have early stage cancer.In some embodiments, the subject has one or more risk factors for cancer, such as tobacco use (e.g., smoking), overweight or obesity, having high body mass index (BMI), being elderly, malnutrition, high alcohol consumption, or family history of cancer.

[0246] In some embodiments, the subject has been using tobacco for at least 1, 5, 10, or 15 years, for example.In some embodiments, the subject has a high BMI, for example, a BMI of 25 or higher, 26 or higher, 27 or higher, 28 or higher, 29 or higher, or 30 or higher.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 is malnourished, for example, has a high consumption of one or more of red meat and / or processed meat, trans fat, saturated fat, and refined sugar, and / or has a low consumption of fruits and vegetables, complex carbohydrates, and / or unsaturated fat. High and low consumption can be defined, for example, as being above or below the recommendations of the Dietary Guidelines for Americans 2020-2025 available at www.dietaryguidelines.gov / sites / default / files / 2021-03 / Dietary_Guidelines_for_Americans-2020-2025.pdf, respectively. In some embodiments, the subject has a high alcohol consumption, e.g., consuming an average of at least 3, 4, or 5 drinks per day (a drink is about 1 ounce or 30 mL of 80 proof distilled spirits or equivalent). In some embodiments, the subject has a family history of cancer, e.g., at least one, two, or three blood relatives have been previously diagnosed with cancer. In some embodiments, relatives are at least the third degree of kinship (e.g., great-grandparents, great aunts or uncles, cousins), at least the second degree of kinship (e.g., grandparents, aunts or uncles, or half-siblings), or first degree of kinship (e.g., parents or siblings).

[0247] In addition, if a cancer is observed to be in remission following treatment, the methods of the invention can be used to monitor for residual disease or recurrence of the disease.

[0248] In some embodiments, the methods and systems disclosed herein can be used to identify individualized or targeted therapies for treating a given disease or condition in a patient based on the presence of one or more proteins of interest and / or classification of nucleic acid variants as somatic or germline in origin. Typically, the disease under consideration is a type of cancer. Non-limiting examples of such cancers include: biliary tract cancer, bladder cancer, transitional cell carcinoma, urothelial carcinoma, brain cancer, glioma, astrocytoma, breast cancer, metaplastic carcinoma, cervical cancer, cervical squamous cell carcinoma, rectal cancer, colorectal cancer, colon cancer, hereditary nonpolyposis colorectal cancer, colorectal adenocarcinoma, gastrointestinal stromal tumor (GIST), endometrial cancer, endometrial stromal sarcoma, esophageal cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, ocular melanoma, uveal melanoma, gallbladder cancer, gallbladder adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma, transitional cell carcinoma, urothelial carcinoma, Wilms' tumor, leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelogenous leukemia (CR ... myelomonocytic leukemia (CMML), liver cancer, hepatoma, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, B cell lymphoma, non-Hodgkin's lymphoma, diffuse large B cell lymphoma, mantle cell lymphoma, T cell lymphoma, non-Hodgkin's lymphoma, precursor T lymphoblastic lymphoma / leukemia, peripheral T cell lymphoma, multiple myeloma, nasopharyngeal carcinoma (NPC), neuroblastoma, oropharyngeal carcinoma, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, pseudopapillary neoplasm, acinar cell carcinoma, prostate cancer, prostate adenocarcinoma, skin cancer, melanoma, malignant melanoma, cutaneous melanoma, small intestine cancer, gastric cancer, gastrointestinal stromal tumor (GIST), uterine cancer, or uterine sarcoma. The type and / or stage of cancer can be detected from genetic alterations including mutations, rare mutations, indels, rearrangements, copy number alterations, transversions, translocations, recombinations, inversions, deletions, aneuploidy, partial aneuploidy, polyploidy, chromosomal instability, chromosomal structural alterations, gene fusions, chromosomal fusions, gene truncations, gene amplification, gene duplications, chromosomal damage, DNA damage, abnormal changes in nucleic acid chemical modifications, abnormal changes in epigenetic patterns, and abnormal changes in nucleic acid 5-methylcytosine.

[0249] Target molecules and genetic data can also be used to characterize specific forms of cancer. Cancers are often heterogeneous in both composition and stage. Genetic profile data can allow characterization of specific subtypes of cancer, which can be important in the diagnosis or treatment of that specific subtype. This information can also provide clues to the subject or attending physician regarding the prognosis of a particular type of cancer, allowing either the subject or attending physician to adapt treatment options according to the progression of the disease. Some cancers can progress and become more aggressive and genetically unstable. Other cancers can remain benign, inactive, or dormant. The systems and methods of the present disclosure can be useful in determining disease progression.

[0250] Furthermore, the method of the present disclosure can be used to characterize the heterogeneity of abnormal conditions in a subject. Such methods can include, for example, generating a genetic profile of extracellular molecules and polynucleotides from a subject, the genetic profile including a plurality of data obtained from copy number variation and rare mutation analysis. In some embodiments, the abnormal condition is cancer. In some embodiments, the abnormal condition can be one that results in a heterogeneous genomic population. In the example of cancer, it is known that some tumors contain tumor cells of cancer at different stages. In other examples, the heterogeneity can include multiple disease foci. Again, in the example of cancer, there can be multiple tumor foci, and perhaps one or more foci are the result of metastasis that has spread from the primary site.

[0251] The method can be used to generate a profile, fingerprint, or dataset that is the sum of target cell and genetic information from different cells of a heterogeneous disease, which may include copy number variation, epigenetic variation, and mutation analysis, either alone or in combination.

[0252] The method can be used to diagnose, prognose, monitor, or observe cancer, precancer, or other disease.In some embodiments, the method herein does not include diagnosing, prognosing, or monitoring fetus, and therefore does not relate to non-invasive prenatal testing.In other embodiments, such methodology can be used in pregnant subjects to diagnose, prognose, monitor, or observe cancer or other disease in fetal subjects where DNA and other polynucleotides may co-circulate with maternal molecules.

[0253] Non-limiting examples of other genetically-based diseases, disorders, or conditions that may be 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) disease, cricketing syndrome, Crohn's disease, cystic fibrosis, Dercum's disease, Down's syndrome, Duane's syndrome, Duchenne muscular dystrophy, factor V Leiden thrombocytosis, familial hypercholesterolemia, familial Mediterranean fever, fragile X syndrome, Gaucher's disease, hemochromatosis, hemophilia, holoprosencephaly, Huntington's disease, Klinefelter's syndrome, Marfan's syndrome, myotonic dystrophy, neurofibromatosis, Noonan's syndrome, osteogenesis imperfecta, Parkinson's disease, phenylketonuria, Poland anomaly ... anomaly), porphyria, progeria, retinitis pigmentosa, severe combined immunodeficiency (SCID), sickle cell disease, spinal muscular atrophy, Tay-Sachs disease, thalassemia, trimethylaminuria, Turner syndrome, palatocardiofacial syndrome, WAGR syndrome, or Wilson's disease.

[0254] In some embodiments, the method described herein comprises detecting the presence or absence of target molecules associated with cell debris originating or derived from tumor cells at a preselected time point after a previous cancer treatment of a subject previously diagnosed with cancer.Also, DNA originating or derived from tumor cells may be detected.The method may further comprise determining a cancer recurrence score indicating the presence or absence of target molecules and, if applicable, DNA originating or derived from tumor cells of the subject.

[0255] When the cancer recurrence score is determined, it can be further used to determine the cancer recurrence status. The cancer recurrence status can be, for example, at risk of cancer recurrence when the cancer recurrence score is above a predetermined threshold. The cancer recurrence status can be, for example, at low or lower risk of cancer recurrence when the cancer recurrence score is above a predetermined threshold. In certain embodiments, a cancer recurrence score equal to a predetermined threshold can result in a cancer recurrence status of either at risk of cancer recurrence or at low or lower risk of cancer recurrence.

[0256] In some embodiments, the cancer recurrence score is compared to a predetermined cancer recurrence threshold, and if the cancer recurrence score is above the cancer recurrence threshold, the subject is classified as a candidate for subsequent cancer treatment, or if the cancer recurrence score is below the cancer recurrence threshold, the subject is classified as not being a candidate for therapy. In certain embodiments, a cancer recurrence score equal to the cancer recurrence threshold may result in the subject being classified as either a candidate for subsequent cancer treatment or not being a candidate for therapy.

[0257] The methods discussed above may further include one or more of any compatible features set forth elsewhere in this specification, including the sections relating to methods for determining a subject's risk of cancer recurrence and / or for classifying a subject as a candidate for subsequent cancer treatment. 2. Methods for determining the risk of cancer recurrence in a subject and / or for classifying a subject as a candidate for subsequent cancer treatment

[0258] In some embodiments, the methods provided herein are methods for determining a subject's risk of cancer recurrence. In some embodiments, the methods provided herein are methods for classifying a subject as a candidate for a subsequent cancer treatment.

[0259] Any of such methods may include collecting a sample from a subject diagnosed with cancer at one or more preselected time points after one or more previous cancer treatments for the subject. The subject may be any of the subjects described herein. The sample may include cell debris. The sample may include DNA, such as cfDNA. The DNA may be obtained from a tissue sample.

[0260] Any such method may include contacting the sample or a subsample thereof with at least one binding molecule and detecting the presence or level of at least one target molecule according to any of the embodiments described herein. Such methods may further include capturing a plurality of sets of target regions from DNA derived from the subject, the plurality of sets of target regions including a set of sequence-variable target regions and / or a set of epigenetic target regions, thereby generating a captured set of DNA molecules. The capturing step may be performed according to any of the embodiments described elsewhere herein. Any such method may include sequencing the captured DNA molecules, thereby generating a set of sequence information. The captured DNA molecules of the set of sequence-variable target regions may be sequenced to a deeper sequencing depth than the captured DNA molecules of the set of epigenetic target regions. Any such method may include using the set of sequence information to detect the presence or absence of DNA originating or derived from tumor cells at a preselected time point. The detection of the presence or absence of DNA originating or derived from tumor cells may be performed according to any of those embodiments described elsewhere herein.

[0261] In any such methods, the previous cancer treatment may include surgery, administration of a therapeutic composition, and / or chemotherapy.

[0262] The method for determining the risk of cancer recurrence of a subject may comprise determining a cancer recurrence score, which indicates the presence or absence or amount of at least one target molecule and / or DNA originating or derived from the subject's tumor cell. The cancer recurrence score can further be used to determine a cancer recurrence status. The cancer recurrence status may be, for example, at risk of cancer recurrence when the cancer recurrence score is above a predetermined threshold. The cancer recurrence status may be, for example, at low or lower risk of cancer recurrence when the cancer recurrence score is above a predetermined threshold. In certain embodiments, a cancer recurrence score equal to a predetermined threshold may result in a cancer recurrence status of either at risk of cancer recurrence or at low or lower risk of cancer recurrence.

[0263] The method for classifying a subject as a candidate for a subsequent cancer treatment may include comparing the subject's cancer recurrence score with a predetermined cancer recurrence threshold, thereby classifying the subject as a candidate for a subsequent cancer treatment if the cancer recurrence score is above the cancer recurrence threshold, or as not a candidate for therapy if the cancer recurrence score is below the cancer recurrence threshold.In certain embodiments, a cancer recurrence score equal to the cancer recurrence threshold may result in the subject being classified as either a candidate for a subsequent cancer treatment or as not a candidate for therapy.In some embodiments, the subsequent cancer treatment comprises the administration of chemotherapy or a therapeutic composition.

[0264] Any such method may include 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, etc.

[0265] In some embodiments, the set of sequence information comprises a sequence variable target region sequence, and determining the cancer recurrence score may comprise determining at least a first subscore indicative of a particular immune cell type, a level of SNVs, insertions / deletions, CNVs, and / or fusions present in the sequence variable target region sequence.

[0266] In some embodiments, the number of mutations in the sequence variable target region selected from 1, 2, 3, 4, or 5 is sufficient to result in a cancer recurrence score in which the first subscore is classified as positive for cancer recurrence. In some embodiments, the number of mutations is selected from 1, 2, or 3.

[0267] In any embodiment in which the Cancer Recurrence Score is classified as positive for cancer recurrence, the subject's Cancer Recurrence Status may be classified as being at risk for cancer recurrence and / or the subject may be classified as being a candidate for subsequent cancer treatment.

[0268] In some embodiments, the cancer is any one of the types of cancer described elsewhere herein, for example, colorectal cancer. 3. Therapy and Related Administration

[0269] In certain embodiments, the methods disclosed herein relate to identifying and administering personalized therapy to a patient. In some embodiments, determining the level of a particular target molecule or cell debris facilitates the selection of an appropriate treatment. In some embodiments, the patient or subject has a given disease, disorder, or condition. Essentially any cancer therapy (e.g., surgery, radiation, and / or chemotherapy, etc.) can be included as part of such methods. In certain embodiments, the therapy administered to the subject includes at least one chemotherapeutic agent. In some embodiments, the chemotherapeutic agent may include alkylating agents (e.g., but not limited to, chlorambucil, cyclophosphamide, cisplatin, and carboplatin), nitrosoureas (e.g., but not limited to, carmustine and lomustine), antimetabolites (e.g., but not limited to, Fluorauracil, methotrexate, and fludarabine), plant alkaloids and natural products (e.g., but not limited to, vincristine, paclitaxel, and topotecan), antitumor antibiotics (e.g., but not limited to, bleomycin, doxorubicin, and mitoxantrone), hormonal agents (e.g., but not limited to, prednisone, dexamethasone, tamoxifen, and leuprolide), and biological response modifiers (e.g., but not limited to, herceptin and avastin, erbitux and rituxan). In some embodiments, the chemotherapy administered to the subject may include FOLFOX or FOLFIRI. In certain embodiments, the subject can be administered a therapy that includes at least one PARP inhibitor. In certain embodiments, the PARP inhibitor can include, among others, olaparib, talazoparib, rucaparib, niraparib (brand name ZEJULA). Typically, the therapy includes at least one immunotherapy (or immunotherapeutic agent). Immunotherapy generally refers to a method for enhancing immune response to a given cancer type. In certain embodiments, immunotherapy refers to a method for enhancing T cell response to tumor or cancer.

[0270] In some embodiments, therapy is individualized based on the status of nucleic acid variants, whether somatic or germline in origin. In some embodiments, essentially any cancer therapy (such as surgery, radiation, and / or chemotherapy) can be included as part of such methods. Typically, individualized therapy includes at least one immunotherapy (or immunotherapeutic agent). Immunotherapy generally refers to a method for enhancing immune response to a given cancer type. In certain embodiments, immunotherapy refers to a method for enhancing T cell response to tumor or cancer.

[0271] In some embodiments, the immunotherapy or immunotherapeutic agent targets immune checkpoint molecules.Certain tumors can evade the immune system by hijacking immune checkpoint pathways.Therefore, targeting immune checkpoints has emerged as an effective approach to combat tumors' ability to evade the immune system and activate antitumor immunity against certain cancers.Pardoll, Nature Reviews Cancer, 2012, 12:252-264.

[0272] In certain embodiments, the immune checkpoint molecule is an inhibitory molecule that reduces signals involved in T cell responses to antigens. For example, CTLA4 is expressed on T cells and plays a role in downregulating T cell activation by binding to CD80 (also known as B7.1) or CD86 (also known as B7.2) on antigen presenting cells. PD-1 is another inhibitory checkpoint molecule expressed on T cells. PD-1 limits the activity of T cells in peripheral tissues during inflammatory responses. In addition, the ligands of PD-1 (PD-L1 or PD-L2) are commonly upregulated on the surface of many different tumors, resulting in downregulation of anti-tumor immune responses in the tumor microenvironment. In certain embodiments, the inhibitory immune checkpoint molecule is CTLA4 or PD-1. In other embodiments, the inhibitory immune checkpoint molecule is a ligand of PD-1, such as PD-L1 or PD-L2. In other embodiments, the inhibitory immune checkpoint molecule is a ligand of CTLA4, such as CD80 or CD86. In other embodiments, the inhibitory immune checkpoint molecule is lymphocyte activation gene 3 (LAG3), killer cell immunoglobulin-like receptor (KIR), T cell membrane protein 3 (TIM3), galectin 9 (GAL9), or adenosine A2a receptor (A2aR).

[0273] Antagonists targeting such immune checkpoint molecules can be used to enhance antigen-specific T cell responses against certain cancers. Thus, in certain embodiments, the immunotherapy or immunotherapeutic agent is an inhibitory immune checkpoint molecule antagonist. 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 inhibitory immune checkpoint molecule antagonist 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-L1, 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-L1 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-L1 antibody, or an anti-PD-L1 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-L1 antibody is one or more of atezolizumab (Tecentriq®), avelumab (Bavencio®), or durvalumab (Imfinzi®).

[0274] In certain embodiments, the immunotherapy or immunotherapeutic agent is an antagonist (e.g., an antibody) against CD80, CD86, LAG3, KIR, TIM3, GAL9, or A2aR. In other embodiments, the antagonist is a soluble form of an inhibitory immune checkpoint molecule, such as a soluble fusion protein comprising the extracellular domain of an inhibitory immune checkpoint molecule and the 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.

[0275] In certain embodiments, the immune checkpoint molecule is a costimulatory molecule that amplifies the signal involved in T cell response to antigen. For example, CD28 is a costimulatory receptor expressed on T cells. When T cells bind to antigen via T cell receptor, CD28 binds to CD80 (also known as B7.1) or CD86 (also known as B7.2) on antigen-presenting cells, amplifying T cell receptor signaling and promoting T cell activation. Because CD28 binds to the same ligands (CD80 and CD86) as CTLA4, CTLA4 can counter or regulate the costimulatory signaling mediated by CD28. In certain embodiments, the immune checkpoint molecule is a costimulatory molecule selected from CD28, inducible T cell costimulator (ICOS), CD137, OX40, or CD27. In other embodiments, the immune checkpoint molecule is a ligand for a costimulatory molecule, including, for example, CD80, CD86, B7RP1, B7-H3, B7-H4, CD137L, OX40L, or CD70.

[0276] Agonists that target these costimulatory checkpoint molecules can be used to enhance antigen-specific T cell responses against certain cancers. Thus, in certain embodiments, the immunotherapy or immunotherapeutic agent is an agonist of a costimulatory checkpoint molecule. In certain embodiments, the agonist of a costimulatory checkpoint molecule is an agonist antibody, preferably a monoclonal antibody. In certain embodiments, the agonist antibody or monoclonal antibody is an anti-CD28 antibody. In other embodiments, the agonist antibody or monoclonal antibody is an anti-ICOS, anti-CD137, anti-OX40, or anti-CD27 antibody. In other embodiments, the agonist antibody or monoclonal antibody is an anti-CD80, anti-CD86, anti-B7RP1, anti-B7-H3, anti-B7-H4, anti-CD137L, anti-OX40L, or anti-CD70 antibody.

[0277] In certain embodiments, the status of nucleic acid variants of a sample from a subject, as being of somatic or germline origin, can be compared with a database of comparator results of a reference population to identify personalized or targeted therapy for the subject.Typically, the reference population includes patients with the same cancer or disease type as the subject, and / or patients who are undergoing or have undergone the same therapy as the subject.Personalized or targeted therapy(s) can be identified when the nucleic acid variants and comparator results meet certain classification criteria (e.g., substantially or nearly identical).

[0278] In certain embodiments, the personalized therapy described herein is typically administered parenterally (e.g., intravenously or subcutaneously). Pharmaceutical compositions that include immunotherapeutic agents are typically administered intravenously. Certain therapeutic agents are administered orally. However, personalized therapy (e.g., immunotherapeutic agents, etc.) can also be administered by any method known in the art, for example, bucally, sublingually, rectally, vaginally, intraurethrally, topically, intraocularly, intranasally, and / or intraauricularly, including by tablet, capsule, granule, aqueous suspension, gel, spray, suppository, salve, or ointment, etc.

[0279] Therapeutic options for treating particular genetically-based diseases, disorders, or conditions other than cancer are generally well known to those of skill in the art and will be apparent given the particular disease, disorder, or condition being considered.

[0280] In some embodiments, for example, when a genetic variant is detected, therapy is personalized based on the status of the nucleic acid variant as being of somatic or germline origin. In some embodiments, essentially any cancer therapy (e.g., surgery, radiation, and / or chemotherapy, etc.) can be included as part of such methods. Typically, personalized therapy includes at least one immunotherapy (or immunotherapeutic agent). Immunotherapy generally refers to a method for enhancing immune response to a given cancer type. In certain embodiments, immunotherapy refers to a method for enhancing T cell response to tumor or cancer.

[0281] In certain embodiments, the status of nucleic acid variants of a sample from a subject as somatic or germline origin can be compared with a database of comparator results of a reference population to identify individualized or targeted therapy for the subject.Typically, the reference population includes patients with the same cancer or disease type as the subject, and / or patients who are undergoing or have undergone the same therapy as the subject.Individualized or targeted therapy(s) can be identified when the nucleic acid variants and comparator results meet certain classification criteria (e.g., substantially or nearly match).

[0282] In certain embodiments, the personalized therapy described herein is typically administered parenterally (e.g., intravenously or subcutaneously). Pharmaceutical compositions that include immunotherapeutic agents are typically administered intravenously. Certain therapeutic agents are administered orally. However, personalized therapy (e.g., immunotherapeutic agents, etc.) can also be administered by methods such as buccal, sublingual, rectal, vaginal, intraurethral, ​​topical, intraocular, intranasal, and / or intraaural, and administration can include tablets, capsules, granules, aqueous suspensions, gels, sprays, suppositories, salves, or ointments, etc. IV. Kit

[0283] Kits comprising the compositions described herein are also provided. The kits can be used in carrying out the methods described herein. In some embodiments, the kits comprise one or more cell debris marker binding molecules. In some embodiments, the kits comprise exosome marker binding molecules, for example, in addition to one or more cell debris marker binding molecules. In some embodiments, the marker binding molecules comprise a label or capture moiety. In some embodiments, the kits comprise a solid support linked to a binding partner of the capture moiety. In some embodiments, the kits comprise one or more target molecule binding molecules. In some embodiments, the kits comprise a reagent for detecting the presence or level of a target molecule.

[0284] In some embodiments, the kit further comprises an agent that recognizes methylcytosine in DNA. In some such embodiments, the agent is an antibody or a methyl-binding protein or domain. In some embodiments, the kit comprises a target-specific probe that specifically binds to a set of epigenetic target regions and / or a set of sequence variable target regions. In some such embodiments, the target-specific probe comprises a capture moiety. In some embodiments, the kit comprises a solid support linked to a binding partner of the capture moiety. In some embodiments, the kit comprises an adaptor. In some embodiments, the kit comprises a PCR primer, where the PCR primer anneals to the target region or the adaptor. In some embodiments, the kit comprises additional elements elsewhere herein. In some embodiments, the kit comprises instructions for carrying out the methods described herein.

[0285] Kit includes ALK, APC, BRAF, CDKN2A, EGFR, ERBB2, FBXW7, KRAS, MYC, NOTCH1, NRAS, PIK3CA, PTEN, RBI, TP53, MET, AR, ABLl, AKTl, ATM, CDHl, CSFIR, CTNNBl, ERBB 4, EZH2, FGFRl, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, MLH1, MPL, NPM1, PDGFRA, PROC, PTPN11, RET, SMAD4, It may further comprise a plurality of oligonucleotide probes selectively hybridizing to at least 5, 6, 7, 8, 9, 10, 20, 30, 40 or all genes selected from the group consisting of SMARCB1, SMO, SRC, STK11, VHL, TERT, CCND1, CDK4, CDKN2B, RAF1, BRCA1, CCND2, CDK6, NF1, TP53, ARID1A, BRCA2, CCNE1, ESR1, RIT1, GATA3, MAP2K1, RHEB, ROS1, ARAF, MAP2K2, NFE2L2, RHOA and NTRK1. The number of genes that the oligonucleotide probes can selectively hybridize to can vary. For example, the number of genes may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54. The kit may include a container comprising a plurality of oligonucleotide probes and instructions for performing any of the methods described herein.

[0286] The kit may include at least 4, 5, 6, 7, or 8 different library adapters with different molecular barcodes and the same sample barcode. The library adapters may not be sequencing adapters. For example, the library adapters do not include flow cell sequences or sequences that allow for the formation of hairpin loops for sequencing. Various variations and combinations of molecular barcodes and sample barcodes are described throughout and are applicable to the kit. Furthermore, in some cases, the adapters are not sequence adapters. In addition, the adapters provided with the kit may further include sequencing adapters. The sequencing adapters may include sequences that hybridize to one or more sequencing primers. The sequencing adapters may further include sequences that hybridize to a solid support, such as flow cell sequences. For example, the sequencing adapters may be flow cell adapters. The sequencing adapters can be attached to one or both ends of the polynucleotide fragments. In some cases, the kit may include at least 8 different library adapters with different molecular barcodes and the same sample barcode. The library adapters may not be sequencing adapters. The kit may further include a sequencing adaptor having a first sequence that selectively hybridizes to the library adaptor and a second sequence that selectively hybridizes to the flow cell sequence. In another example, the sequencing adaptor may be hairpin shaped. For example, the hairpin shaped adaptor may include a complementary double-stranded portion and a loop portion, and the double-stranded portion may be attached (e.g., ligated) to a double-stranded polynucleotide. The hairpin shaped sequencing adaptor may be attached to both ends of a polynucleotide fragment to generate a circular molecule that is sequenced multiple times. The sequencing adaptor may include one or more barcodes. For example, the sequencing adaptor may include a sample barcode. The sample barcode may include a predetermined sequence. The sample barcode may be used to identify the source of the polynucleotide.The sample barcode may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more (or any length described throughout) nucleobases, e.g., at least 8 bases. As described above, the barcode may be a continuous or discontinuous sequence.

[0287] The library adaptors may be blunt ended and Y-shaped and may be less than or equal to 40 nucleobases in length. Other variations of library adaptors can be found throughout and are applicable to the kit.

[0288] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific examples provided herein. Although the present invention has been described with reference to the above specification, the description and illustration of the embodiments herein are not intended to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the present invention. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific illustrations, configurations, or relative proportions shown herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives to the disclosed embodiments described herein can be used in the practice of the present invention. It is therefore contemplated that the present disclosure will cover any such alternatives, modifications, variations, or equivalents. It is intended that the scope of the present invention is defined by the following claims, and that methods and structures within the scope of such claims and their equivalents are covered thereby.

[0289] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those skilled in the art upon reading this disclosure that various changes in form and detail may be made therein without departing from the true scope of the disclosure and may be practiced within the scope of the appended claims. For example, the methods, systems, computer readable media, and / or component features, steps, elements, or other aspects thereof may all be used in various combinations.

[0290] All patents, patent applications, websites, other publications or documents, and accession numbers, etc. cited herein are incorporated by reference in their entirety for all purposes as if each individual item was specifically and individually indicated to be incorporated by reference. Where different versions of sequences are associated with accession numbers at different times, the version associated with the accession number at the effective filing date of this application is intended. By effective filing date, we mean the earlier of the actual filing date or, if applicable, the filing date of the priority application referencing the accession number. Similarly, where different versions of a publication or website, etc., were published at different times, the latest published version at the effective filing date of this application is intended unless otherwise indicated. EXAMPLES

[0291] Example 1 Analysis of circulating proteins using simultaneous detection of target proteins and exosome markers A set of patient samples is analyzed by a blood-based assay to detect the presence / absence of cancer. A first portion of the whole blood sample of such a patient is fractionated into plasma, buffy coat, and red blood cell fractions by centrifugation at 1,600g for 10 minutes at 10°C. The plasma fraction is further fractionated by centrifugation at 3,220g for 10 minutes at 10°C to generate a plasma pellet and a plasma supernatant. The buffy coat fraction, the plasma pellet, and a second portion of the whole blood sample are contacted with a first antibody conjugated to a first oligonucleotide that specifically binds to phosphatidylserine, and a second antibody conjugated to a second oligonucleotide that specifically binds to a target molecule, such as CTLA4 or PDL1. Optionally, the plasma supernatant is contacted with a first antibody conjugated to a first oligonucleotide that specifically binds to an exosome marker, and a second antibody conjugated to a second oligonucleotide that specifically binds to a target molecule, such as CTLA4 or PDL1. Each of the first and second oligonucleotides comprises two parts: 1) a first part with a sequence (molecular barcode) that is unique to the antibody being conjugated, and 2) a second part that is 3' to the first part with a hybridization sequence. The hybridization sequences of the first oligonucleotides are the same as each other and are complementary to the hybridization sequence of each second oligonucleotide. The first and second oligonucleotides hybridize when they are within close enough proximity to each other, and the hybridized (double-stranded) oligonucleotide sequence is extended from the 3' end of the hybridization sequence using DNA polymerase. The extended oligonucleotides are pooled, amplified, and sequenced using an Illumina sequencer or quantified by a suitable procedure such as qPCR. For an exemplary hybridization, extension, and sequencing-based detection procedure, see, for example, Gong et al., Bioconjugate Chem. 2016, 27, 1, 217-225.

[0292] The sequence reads generated by the sequencer are then analyzed using bioinformatics tools / algorithms. The molecular barcodes present in the sequenced molecules are used to identify the antibodies and their antigen molecules, and also to deconvolute the cell debris or exosome markers to which they are in close proximity, if applicable. Quantification of sequence reads corresponding to proteins, such as proteins upregulated in tumor cells, in samples from patients compared to samples from healthy subjects facilitates the determination of the likelihood that the patient has cancer. Example 2 Analysis of circulating proteins using sequential detection of target proteins and exosome markers

[0293] A set of patient whole blood samples is fractionated as described in Example 1. The buffy coat fraction, plasma pellet, and a second portion of the whole blood sample are contacted with Annexin V conjugated to biotin and then with magnetic beads conjugated to streptavidin to isolate cellular debris. Optionally, the plasma supernatant is contacted with an exosome marker binding molecule conjugated to biotin and then with magnetic beads conjugated to streptavidin to isolate exosomes. The beads and their bound molecules are precipitated and any unbound sample components are washed off the beads with buffers containing increasing concentrations of salt. Cell debris is washed off Annexin V using a high salt buffer, and exosomes are washed off the exosome marker binding molecule, if applicable. The precipitated and enriched molecules are purified to remove salt and concentrated in preparation for the target molecule detection step.

[0294] If the identity of the target molecule is known, the isolated cell debris and exosomes are contacted with an antibody to the target molecule, if applicable, and detected using immunoassays or flow cytometry. If the identity of the target molecule is known or unknown, if applicable, the proteins are purified from the membranes and other material of the cell debris and exosomes, and the proteins are analyzed by mass spectrometry to identify and / or quantitate them. Example 3 Combined analysis of circulating proteins and cfDNA using sequential detection of target proteins and exosomal markers

[0295] A set of patient whole blood samples is fractionated as described in Example 1. The buffy coat fraction, plasma pellet, and a second portion of the whole blood sample are processed and analyzed as described in Example 2. The plasma supernatant is divided into multiple aliquots. The first aliquot is processed and analyzed as described in Example 2. cfDNA is extracted from the second aliquot.

[0296] Then, the cfDNA of the subject sample is divided according to the cytosine methylation level.The cfDNA is contacted with an antibody that recognizes methylcytosine, and then immunoprecipitated using magnetic beads that are conjugated with protein G, thus dividing into highly methylated DNA and low methylated DNA.Any DNA that is not methylated or is low in methylation is washed out from the beads using a buffer that contains increasing concentrations of salt.Finally, a high salt buffer is used to wash out highly methylated DNA from the antibody, providing a highly methylated distribution fraction, an intermediate distribution fraction, and a low methylated distribution fraction.

[0297] After the cfDNA of the distribution fraction is concentrated, the first adaptor is added to the cfDNA by ligation to its 3' end. The adaptor is used as a priming site for second strand synthesis using a universal primer and DNA polymerase. The first adaptor comprises biotin, and the nucleic acid ligated to the first adaptor is bound to beads that comprise streptavidin. Then, the second adaptor is ligated to the 3' end of the second strand of the now double-stranded molecule. These adaptors comprise non-unique molecular barcodes, and each distribution fraction is ligated to an adaptor that has a non-unique molecular barcode that is distinguishable from the barcodes of the adaptors used in other distribution fractions. After ligation, the distribution fractions are pooled together and amplified by PCR.

[0298] After PCR, the amplified DNA is washed and concentrated before enrichment. Once concentrated, the amplified DNA is combined with a salt buffer and a biotinylated RNA probe comprising a probe for a set of sequence variable target regions and a probe for a set of epigenetic target regions, and the mixture is incubated overnight. The probe for the set of sequence variable target regions has a footprint of about 50 kb, and the probe for the set of epigenetic target regions has a footprint of about 500 kb. The probe for the set of sequence variable target regions comprises oligonucleotides that target at least a subset of the genes described herein, and the probe for the set of epigenetic target regions comprises oligonucleotides that target one or more of a hypermethylated variable target region, a hypomethylated variable target region, and optionally a CTCF binding target region, a transcription start site target region, a local amplification target region, and a methylation control region.

[0299] Biotinylated RNA probes (hybridized to DNA) are captured with streptavidin magnetic beads and separated from non-captured amplified DNA by a series of salt-based washes, thereby enriching the sample. After enrichment, an aliquot of the enriched sample is sequenced using an Illumina NovaSeq sequencer. The sequence reads generated by the sequencer are then analyzed using bioinformatics tools / algorithms. Molecular barcodes are used to identify unique molecules and deconvolute the sample into differentially distributed molecules. The method described in this example, apart from providing information on the overall methylation level of molecules based on their distribution (i.e., methylated cytosine residues), can also provide higher resolution information on the identity and / or location of the type of methylated cytosine. The sequence-variable target region sequences are analyzed by detecting genomic alterations such as SNVs, insertions, deletions, and fusions that can be called with sufficient certainty to distinguish actual tumor variants from technical errors (e.g., PCR errors, sequencing errors). Epigenetic target region sequences are analyzed independently to detect methylated cfDNA molecules in regions shown to be differentially methylated in cancer compared to normal cells. Finally, the results of the analyses are combined to generate a final tumor presence / absence call.

Claims

Claim 1 A method for detecting a cell debris-associated target molecule in a sample, comprising: a) contacting the sample or a sub-sample thereof with at least one binding molecule, wherein the at least one binding molecule binds to a cell debris marker, thereby generating a complex comprising the at least one binding molecule and cell debris, wherein the cell debris comprises membrane fragments; and b) detecting the presence or level of at least one target molecule associated with the complex. A method comprising the above steps. Claim 2 The sample is obtained from a subject and / or the sample is a blood sample, for example, the blood sample is: i) a whole blood sample; ii) a plasma sample; or iii) a plasma pellet sample or a buffy coat sample. The method according to claim 1. Claim 3 (i) The at least one binding molecule is a protein, which is an antibody if necessary, and / or ii) the at least one binding molecule binds to a cell debris marker, for example, the cell debris marker is an inner membrane marker, and / or the cell debris marker is phosphatidylserine or phosphatidylethanolamine, for example: a) the cell debris marker is phosphatidylserine, and the at least one binding molecule is annexin V or an antibody specific for phosphatidylserine; or b) the cell debris marker is phosphatidylethanolamine, and the at least one binding molecule is an antibody specific for phosphatidylethanolamine. The method according to claim 1 or claim 2. Claim 4 The at least one binding molecule comprises a label or is conjugated to a solid support, for example: i) the at least one binding molecule comprises a label, and the method further comprises capturing the at least one binding molecule by binding the label to a solid support; ii) the at least one binding molecule is conjugated to a label, and the label comprises a fluorophore, biotin, peptide, or oligonucleotide; and / or iii) the solid support comprises beads, for example, the at least one binding molecule is conjugated to magnetic beads. The method according to claim 1. Claim 5 Before the detecting, capturing the complex from the sample or a sub-sample thereof, for example, the capturing includes separating components of the sample or the sub-sample thereof that are not bound to the at least one binding molecule from the complex to which the at least one binding molecule is bound. The method according to claim 1.

6. The detecting includes mass spectrometry of a target molecule associated with the complex. The method according to claim 5.

7. The detecting includes contacting the complex with at least one binding molecule that binds to a target molecule that may associate with the complex. For example, the at least one binding molecule that binds to a target molecule associated with the complex is an antibody specific for the target molecule. The method according to claim 5.

8. At least one binding molecule that binds to a target molecule includes a label. For example, the label is a fluorophore or an oligonucleotide. The method according to claim 7.

9. The label is an oligonucleotide. For example, the label is an oligonucleotide, the binding molecule that binds to a cell debris marker includes an oligonucleotide, and optionally, the detecting includes a proximity ligation assay or a proximity extension assay. The method according to claim 8.

10. The detecting (i) includes an immunoassay. For example, the immunoassay is an enzyme-linked immunosorbent assay, a sandwich assay, an electrochemiluminescence assay, or a multiplex immunoassay, and / or (ii) includes flow cytometric analysis of the complex. The method according to any one of claims 5 to 9.

11. A plurality of target molecules associated with the complex are detected. For example, the plurality of target molecules are 2 to 10,000, 2 to 5,000, 2 to 1,000, or 2 to 100 target molecules. The method according to claim 1.

12. The method according to claim 1, including capturing the complex after contacting the complex with at least one binding molecule that binds to a target molecule that may associate with the complex. **Claim 13** (i) At least one target molecule, two or more of said plurality of target molecules, or each of said plurality of target molecules is a protein, and / or at least one target molecule, two or more of said plurality of target molecules, or each of said plurality of target molecules is a carbohydrate, optionally a glycoprotein carbohydrate. (ii) At least one target molecule is a molecule associated with a disease, two or more of said plurality of target molecules are molecules associated with a disease, or each of said plurality of target molecules is a molecule associated with a disease. For example, (a) said disease is cancer, for example, said at least one target molecule is upregulated in tumor cells compared to healthy cells of the same tissue type, and / or (b) at least one, two or more, or each of said target molecules is selected from PD-L1, CTLA4, NY-ESO1, mesothelin, CA15-3, CA19-9, CA-125, and CA-172-4, and / or (iii) At least one target molecule, two or more target molecules, or each of said plurality of target molecules is a cell type marker, optionally said cell type marker is selected from markers of immune cells and solid tissue cells, and further optionally said cell type marker is selected from markers of colon, lung, breast, skin, prostate, stomach, pancreas, and hepatocyte type markers. The method according to claim 11. **Claim 14** The method according to claim 1, wherein said sample is obtained from a subject having a disease, said detecting comprises identifying a plurality of target molecules, and said identifying comprises mass spectrometry of the target protein. **Claim 15** (i) Comprising measuring the total cell debris level in said sample or a sub-sample thereof. For example, said sample is obtained from a subject having a disease, and said total cell debris level is measured relative to the total cell debris level in a sample or a sub-sample obtained from a healthy individual, and / or (ii) analyzing DNA in a sub-sample of the sample or in a second sample obtained from the same subject from which the first sample was obtained, wherein, optionally, the sub-sample or the second sample is a plasma sample or a serum sample, and further optionally, the DNA is cfDNA; The method according to claim 1.

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