Compositions and methods for identifying cell types

Abstract

The present disclosure relates generally to compositions and methods for determining cell types based on methylation profiles of associated DNA. For cell-free DNA, this determination can be used to identify diseases or conditions associated with cell types. For tumor cells, this determination is used to identify their primary origin.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 295,319, filed Dec. 30, 2021, under 35 U.S.C. § 119(e), the content of which is hereby incorporated by reference in its entirety.

[0003] The Electronic Sequence Listing Mentioned

[0004] The content of the electronic sequence listing (334655WO.xml; size: 13,761,379 bytes; created date: Dec. 27, 2022) is hereby incorporated by reference in its entirety. BACKGROUND OF THE INVENTION

[0005] Identifying the source of cellular or cell-free DNA has important implications. For example, tumor cells may migrate to other tissues, making it challenging to identify their origin. Cancer of unknown primary (CUP) is cancer that is judged to be in a metastatic stage at the time of diagnosis, but the primary tumor cannot be identified. Approximately 3% to 5% of all people diagnosed with invasive cancer are found to have CUP, and in most (80% to 85%) of these cases, the prognosis is poor.

[0006] Small fragments of nucleic acids (e.g., DNA) circulate freely in the peripheral blood of healthy and diseased individuals. These cell-free nucleic acids, such as DNA (cfDNA) molecules, may be derived from dying or damaged cells and thus reflect ongoing cell death or injury occurring in the body. In recent years, this understanding has led to the emergence of diagnostic tools that impact multiple medical fields. For example, next-generation sequencing of fetal DNA circulating in maternal blood has allowed non-invasive prenatal testing for fetal chromosomal abnormalities; detecting donor-derived DNA circulating in organ transplant recipients can be used for early identification of transplant rejection; and, evaluating circulating mutant DNA can be used to detect genotypes and monitor cancer.

[0007] Such techniques are very effective in identifying genetic abnormalities in circulating DNA or displaced cells, but do not provide information when the DNA does not carry mutations. A major limitation of sequencing is that it does not reveal the tissue source of the DNA, thus hindering the identification of tissue-specific cancers or cell death. The latter is crucial in many contexts that do not involve DNA mutations, such as neurodegenerative diseases, inflammatory diseases, or ischemic diseases. Even in oncology, in addition to determining the mutational profile of a tumor, determining the tissue source of the tumor is often important, for example in CUP, and in the context of early cancer diagnosis.

[0008] Identifying the tissue origin of DNA can also provide insights into collateral tissue damage (e.g., the toxicity of a drug in genetically normal tissue), which is a key element in drug development and monitoring treatment response. SUMMARY OF THE INVENTION

[0009] The present disclosure provides compositions and methods for determining cell type based on the methylation status of DNA fragments. Compositions and methods are also provided for identifying such diseases and conditions by cell-free DNA released by cells affected by a disease or condition in a subject (e.g., a human subject). In oncology, or in another disease state, the techniques of the present invention can be used to identify the primary source of tumor cells.

[0010] In one embodiment, the present disclosure provides a method for identifying that a biological sample comprises DNA from a cell type. In some embodiments, the cell type is selected from the group consisting of: oral, laryngeal, and esophageal epithelium, gastric epithelium, small intestinal epithelium, colonic epithelium, colonic fibroblasts, gallbladder epithelium, liver hepatocytes, pancreatic acinar cells, pancreatic alpha cells, pancreatic beta cells, pancreatic delta cells, pancreatic ductal cells, endometrial epithelium, fallopian tube epithelium, renal epithelium, bladder epithelium, prostatic epithelium, mammary basal epithelium, mammary luminal epithelium, alveolar epithelium, lung bronchial epithelium, heart cardiomyocytes, heart fibroblasts, vascular endothelial cells, blood b cells, blood granulocytes, blood monocytes + macrophages, blood natural killer cells, blood t cells, erythroid progenitors, epidermal keratinocytes, dermal fibroblasts, osteoblasts, skeletal muscle cells, smooth muscle cells, thyroid epithelium, adipocytes, neuronal CNS, and oligodendrocytes.

[0011] In some embodiments, the method requires detecting the methylation status of each of at least four, or at least five, six, seven, or eight CpG sites of a target DNA fragment in a biological sample, and then identifying the target DNA fragment as being from the human cell type when the methylation status of the target DNA fragment corresponds to the methylation status of the DNA fragment defined for a human cell type in Table A.

[0012] As used herein, in some embodiments, the methylation status refers to the percentage of CpG sites within a target DNA fragment that are methylated (e.g., 25%). In some embodiments, the methylation status refers to whether the target DNA fragment is hypermethylated (M, at least 60% CpG methylation) or hypomethylated (U, no more than 40% CpG methylation) compared to the same fragment in other cell types.

[0013] In some embodiments, the target DNA fragment has the DNA sequence as shown in Schedule B and the Sequence Listing. However, as demonstrated in the experimental examples, the methylation pattern is consistent over contiguous regions. Thus, these sequences or their genomic positions represent nearby genomic regions.

[0014] In some embodiments, the target DNA fragment is a DNA fragment that includes at least one CpG site within the sequence included in the Sequence Listing. In some embodiments, the target DNA fragment is a DNA fragment that includes at least two CpG sites within the sequence included in the Sequence Listing. In some embodiments, the target DNA fragment is a DNA fragment that includes at least three or four CpG sites within the sequence included in the Sequence Listing.

[0015] In some embodiments, the target DNA fragment is located at a position less than 1000 bp from the 5' end or 3' end of the sequence included in the Sequence Listing. In some embodiments, the target DNA fragment is located at a position less than 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 400 bp, 300 bp, 250 bp, 200 bp, or 150 bp from the 5' end or 3' end of the sequence included in the Sequence Listing.

[0016] In some embodiments, the target DNA fragment is obtained from a biological sample selected from the group consisting of blood, plasma, serum, semen, milk, urine, saliva, and cerebrospinal fluid.

[0017] In some embodiments, the target DNA fragment is a cell-free DNA fragment. In some embodiments, identifying the cell-free DNA fragment as being from a cell type includes detecting abnormal cell death of that cell type, or a disease associated with that cell type. In some embodiments, the method further requires identifying that a human subject has or may have damage, inflammation, or cancer in the corresponding cell type.

[0018] In some embodiments, the disease or condition is a physical injury, inflammation, infection, cancer, diabetes, autoimmune disease, multiple sclerosis (MS), or a neurodegenerative disorder.

[0019] In some embodiments, the length of the target DNA fragment is from 20 bp to 500 bp. In some embodiments, the length of the target DNA fragment is from 30 bp to 400 bp, 40 bp to 300 bp, 50 bp to 250 bp, 50 bp to 200 bp, or 50 bp to 150 bp, but is not limited thereto.

[0020] In some embodiments, the methylation state is the conversion of cytosine to 5-methylcytosine (5-mC) or 5-hydroxymethylcytosine (5-hmC). In some embodiments, detecting the methylation state includes treating the DNA fragment with bisulfite or an enzyme, or digesting the DNA fragment with a restriction endonuclease sensitive to DNA methylation. In some embodiments, the enzyme treatment includes treating with APOBEC-Seq. In some embodiments, detecting the methylation state further includes determining the sequence of the DNA fragment. In some embodiments, the sequence is determined by deep sequencing.

[0021] In some embodiments, the method also detects genetic variations in the target DNA fragment, thereby determining that the cells releasing the target DNA fragment contain the genetic variation. In some embodiments, the method further includes administering to the patient an agent that can be used to treat the identified disease or condition. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The methylation profiles of the adult body are presented. 207 healthy samples were obtained from adults, isolated and deep sequenced (WGBS, average depth >30x) to form a comprehensive human cell type-specific methylation profile.

[0023] Figure 2 Shows the human genome partitioned into 7,264,350 contiguous homogeneous blocks. These histograms show the number of segmented blocks as a function of their length in bases (left), or as a function of the number of CpGs they contain (right). In addition to 2,746,623 blocks of length 3 to 30 CpGs (plotted as above), there are also 3,271,607 blocks containing one CpG and 1,185,719 blocks containing two CpGs, and 60,401 blocks containing more than 30 CpGs.

[0024] Figure 3 Shows biological replicates of the same cell type from different individuals, showing a surprisingly low ratio of differentially methylated blocks. This focuses on 37 cell subtypes with n≥3 replicates (e.g., endothelial cells from a specific tissue), and the average percentage (shown on the Y-axis) of methylated blocks (≥3 CpGs) with a 50% difference in methylation degree (absolute Δβ) measured across replicates. The blocks of almost all cell subtypes (36 / 37) differ by ≤0.5%, indicating a very high degree of conservation among replicates. The red dashed line marks the average number of different blocks (4.9%) between two random samples of different cell types.

[0025] Figure 4 Shows an unsupervised agglomerative hierarchical clustering reflecting the human developmental lineage of healthy cell types.

[0026] Figure 5 Shows the average methylation in the top-ranked differentially methylated blocks. Shows the average methylation values at the 1% most variable blocks (21,077 blocks) containing more than 4 CpGs. For each block, we calculated the average methylation in each sample and classified it as unmethylated (<50%) or methylated (>50%). The box plots show the 25th to 75th percentiles among the average methylation levels of unmethylated blocks / samples (blue), methylated blocks (yellow), or the difference between methylated and unmethylated samples in the same block (green).

[0027] Figure 6 Shows the human methylation profiles of 207 samples across 39 cell types. (A) 953 genomic regions that are unmethylated in a cell type-specific manner. Each cell in the chart is labeled with the average methylation of one genomic region (column) in each of the 39 cell types (rows). Each cell type shows up to 25 regions with an average length of 251 bp (9 CpGs) each. (B) The top 25 cardiomyocyte regions. For each region, the average methylation of each CpG site (column) in all 207 samples is plotted in the profile and grouped into 39 cell types as described above. (C) A locus specifically unmethylated in cardiomyocytes. This marker (highlighted in light blue) is 120 bp long (6 CpGs) and is located in the first intron of MYL4, a heart-specific gene (TPM expression of 2518 in atrial appendages, GTEx inset). The genomic snapshot depicts the average methylation (purple trace) of six cardiomyocyte samples, four cardiac fibroblast samples, and three aortic samples (two endothelial cells, one smooth muscle cell). (D) Visualization of bisulfite-converted fragments from three cardiomyocyte samples, one cardiac fibroblast sample, and two aortic samples (endothelial and smooth muscle). Reads mapped to chr17:45289451 - 45289570 (hg19) that cover at least 3 CpGs are shown. Yellow / blue dots depict methylated / unmethylated CpG sites.

[0028] Figure 7 Shows the enrichment of cell type-specific marker regulatory motifs. Using HOMER motif analysis, the top-ranked transcription factor binding site motifs that are enriched among the top 250 differentially unmethylated regions in each cell type are shown. Motifs similar to previous (more significant) hits are skipped.

[0029] Figure 8Cell type-specific hypermethylated regions are shown to be enriched for CpG islands, Polycomb targets, and CTCF and REST / NSRF. (A) The top 37.9% of cell type-specific hypermethylation markers (1,185 out of 3,125 markers, p < 1E-100) overlap with CpG islands. In comparison, 1.7% of cell type-specific hypomethylated regions (198 / 11,371, p < 2E-29) overlap with CpG islands, which constitute <0.9% of the genome (black line). (B) These regions are generally enriched for H3K27me3 in other cell types. Shown are the average H3K27me3 signals in monocytes and macrophages near all cell type-specific hypermethylated regions (top, blue) or near monocyte / macrophage-specific hypermethylated regions (green). (C) Similar plots of Polycomb annotation (chromHMM) in monocytes and macrophages for all markers or monocyte / macrophage-specific markers. (D) Motif analysis of cell type-specific hypermethylated regions (top 100 for each cell type) identifies known CTCF and REST / NSRF motifs. (E) Results of ChIP-seq data analysis for one such locus (chr1:209364093-209364250, highlighted in blue, hg19), which is specifically methylated in small intestine and colon epithelium (box 1) and unmethylated elsewhere. As shown below, this locus binds in multiple cell types and tissues but is mostly unbound in vivo in stomach and colon epithelium (box 2). (F) Based on REST target expression in the endocrine pancreas, the REST / NSRF motif is present in 15 out of the top 100 (15%) cell type-specific hypermethylated regions in the endocrine pancreas (α, β, and δ cells), in 5 out of the top 100 pancreatic δ cells, and in 2 out of the top 100 pancreatic β cells, compared to ~0.1% in background sequences.

[0030] Figure 9Show the results of lung epithelial methylome analysis. A. Tissue methylome analysis for comparison revealed multiple methylation blocks as follows: methylation blocks uniquely unmethylated in alveoli (1,663 blocks), bronchial epithelial cells (673 blocks), or both (139 blocks), and methylation blocks methylated in all other tissues. Another 11 markers specifically methylated in the lung are not shown. Each marker covers ≥3 CpGs and exhibits an average methylation increment of ≥0.4 between the 25th percentile of the target cell type and the 97.5th percentile of other tissues. B. Characterize an alveoli-specific methylation marker located at chr16:667119-667272 (hg19) in the Rab40C gene. This region is unmethylated only in alveolar epithelium and is enriched for chromatin markers H3K27ac, H3K4mel, and H3K4me3. C. Lung-specific methylation markers are enriched in enhancer regions. For each of the three marker groups, the number of markers with enhancer-related chromatin states in the lung is shown, indicating an enrichment change of 2.5- to 10-fold. D. GREAT annotation, identifying gene sets enriched among genes closest to lung-unique methylation markers. The 5 most significant (BinomFDRQ) gene sets in the methylation markers of each lung cell type are shown.

[0031] Figure 10 Show the performance of selected lung-specific markers. A. Determination of specificity. Methylation status of lung epithelial markers (green for alveoli, orange for bronchi, pink for normal lung) in DNA from multiple tissues. The percentage of molecules with most CpG sites methylated or unmethylated is shown. B. Determination of specificity in lung cancer. Methylation status of lung epithelial markers in DNA from multiple lung cancers. The percentage of molecules with CpG sites of this marker methylated or unmethylated is shown. This analysis is based on TCGA Illumina BeadCheap array data, where each locus is represented by one CpG site. Note that lung cancer retains the methylation pattern of normal lung. C. Determination of sensitivity and accuracy in vitro. Mix DNA from healthy human alveolar (left) or bronchial (right) epithelium with blood DNA as indicated, and then determine the fraction of methylated or unmethylated molecules in the lung markers. D. Determination of robustness. Analyze lung markers in cfDNA samples extracted from the same donor in parallel. The number of genome equivalents per ml of plasma present in each parallel sample is shown.

[0032] Figure 11Shows the detection results of lung-derived cfDNA in healthy individuals. A. Concentrations of lung cfDNA in plasma from 30 healthy donors. Concentrations were measured by multiplying the fraction of lung cfDNA by the total cfDNA concentration. B. Fractions of lung cfDNA in plasma from 30 healthy donors and in bronchoalveolar lavage fluid from 6 donors.

[0033] Figure 12 Shows the identification of lung-derived cfDNA in lung cancer patients. A. Lung cfDNA in plasma from 26 advanced lung cancer patients. Concentrations were measured by multiplying the fraction of lung cfDNA by the total cfDNA concentration. The dashed lines in this panel and panel C represent the mean + 2 standard errors of healthy controls. B. Lung cfDNA in plasma from lung cancer patients. Top, P values determined by two-tailed Mann-Whitney test. Bottom, ROC curve for all advanced lung cancer patients versus healthy samples. C. Lung cfDNA in plasma from 51 donors undergoing bronchoscopy. Concentrations were measured by multiplying the fraction of lung cfDNA by the total cfDNA concentration. P values determined by two-tailed Mann-Whitney test. Left, each color represents the cumulative value of markers for the specified cell type. Right, each point represents the cumulative value of all lung markers measured. D. Concentrations of lung cfDNA in plasma from donors undergoing bronchoscopy versus healthy patients (left), and ROC curve for differentiating patients with pulmonary conditions from healthy controls.

[0034] Figure 13 Shows the effect of the number of lung markers on assay sensitivity. A. ROC curves using the specified combinations of lung methylation markers for differentiating patients with any pulmonary condition from healthy controls. B. Sensitivity at 70% specificity for the specified combinations of lung markers. Patients with pulmonary conditions versus healthy controls.

[0035] Figure 14 Shows the detection results of lung-specific cfDNA in COPD patients. A. Concentrations of lung cfDNA in plasma from 77 COPD patients. Concentrations were measured by multiplying the fraction of lung cfDNA by the total cfDNA concentration. The dashed line represents the mean + 2 standard errors of healthy controls. B. Lung cfDNA in plasma from lung cancer patients, exacerbation and stable-phase COPD patients, and healthy controls. C. Lung cfDNA in plasma from COPD patients who were still alive 14 months after sampling versus patients who died during this period.

[0036] Figure 15 Is a schematic diagram showing computing components that can be used to implement various features of the embodiments described in this disclosure. Detailed Description

[0037] The following description sets forth exemplary embodiments of the technology of the present invention. However, it should be recognized that such a description is not intended as a limitation on the scope of the present disclosure, but rather is provided as a description of exemplary embodiments.

[0038] Definitions

[0039] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this specification pertains. As used herein, the following terms have the meanings given to them below.

[0040] As used herein, the term "methylation" refers to the process by which a methyl group is attached to a nucleic acid (e.g., a DNA molecule). For example, a hydrogen atom on the pyrimidine ring of a cytosine base can be converted to a methyl group, thereby forming 5-methylcytosine. The term also includes the process by which a hydroxymethyl group is attached to a DNA molecule (specifically "hydroxymethylation"), e.g., by oxidizing the methyl group on the pyrimidine ring of a cytosine base. Methylation (including hydroxymethylation) typically occurs at dinucleotides of cytosine and guanine, herein referred to as "CpG dinucleotides" or "CpG sites". The principles described herein also apply to the detection of methylation in non-CpG contexts, including non-cytosine methylation. In such embodiments, the wet-lab assays for detecting methylation may differ from any of the assays described herein. Additionally, a methylation status vector may contain elements of a vector that are typically sites where methylation has occurred or not occurred (even if those sites are not specifically CpG sites).

[0041] As used herein, the term "methylation site" refers to the region of a DNA molecule at which a methyl group can be attached to the DNA molecule. A "CpG" site is the most common methylation site, but methylation sites are not limited to CpG sites. For example, DNA methylation may occur in cytosines of CHG and CHH, where H is adenine, cytosine, or thymine.

[0042] As used herein, the term "CpG site" refers to the region of a DNA molecule at which, in the linear base sequence along its 5' to 3' direction, a cytosine nucleotide is followed by a guanine nucleotide. "CpG" is an abbreviation for 5'-C-phosphate-G-3', i.e., cytosine and guanine are separated by only one phosphate group. The cytosine in a CpG dinucleotide can be methylated to form 5-methylcytosine.

[0043] As used herein, the terms "hypomethylation" or "hypermethylation" refer to the following methylation states of DNA molecules containing multiple CpG sites (e.g., more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, or more than 10, etc.): in which, compared to the corresponding DNA molecules from one or more reference samples, a higher percentage of CpG sites (e.g., more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 40%, more than 50%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 97.5%, more than 98%, more than 99%, more than 99.9%, or any other numerical percentage within the range of 0% to 50% or 50% to 100%, where each range provided in this disclosure includes the range limit endpoints, e.g., 50% and 100%) are either unmethylated or methylated. In the context of cancer, the reference sample can be normal tissue. Hypomethylation of a DNA molecule from a tumor cell means a decreased methylation percentage compared to normal tissue (e.g., healthy non-diseased tissue (e.g., non-cancerous tissue)), also referred to as "hypomethylation". A "hypomethylated" nucleic acid (e.g., cfDNA) fragment can be a fragment having a number (e.g., more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, or more than 10) of CpG sites and a percentage (e.g., more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 97.5%, more than 98%, more than 99%, more than 99.9%) of these CpG sites being unmethylated. Hypermethylation of a DNA molecule from a tumor cell means an increased methylation percentage compared to normal tissue (e.g., healthy non-diseased tissue (e.g., non-cancerous tissue)), also referred to as "hypermethylation". Similarly, a "hypermethylated" nucleic acid (e.g., cfDNA) fragment can be a fragment having a number (e.g., more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, or more than 10) of CpG sites and a percentage (e.g., more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 97.5%, more than 98%, more than 99%, more than 99.9%) of these CpG sites being methylated. "Hypomethylation" can also refer to a lower methylation percentage of a DNA molecule in a target cell compared to other types of cells, while "hypermethylation" can also refer to a higher methylation percentage of a DNA molecule in a target cell compared to other types of cells.

[0044] The term "cell-free nucleic acid" refers to fragments of the following nucleic acids (e.g., DNA in the form of "cell-free DNA" and "cfDNA") that circulate in an individual's body (e.g., bloodstream) and are derived from one or more healthy cells and / or from one or more diseased, aging, or damaged cells. In addition, cell-free nucleic acids (such as cfDNA) can be derived from other sources, such as viruses, fetuses, etc.

[0045] The terms "circulating tumor DNA" and "ctDNA" refer to the following DNA fragments that are derived from tumor cells and can be released into an individual's bloodstream as a result of biological processes such as apoptosis or necrosis of dying cells, or actively released by living tumor cells.

[0046] As used herein, the terms "abnormal methylation pattern" and "anomalous methylation pattern" refer to the following methylation patterns of nucleic acid (e.g., DNA, such as cfDNA) molecules or methylation state carriers that are found and / or expected to be found in a sample at a lower frequency than would be found and / or expected to be found in a healthy (e.g., non-cancer) sample. In various embodiments, such methylation patterns are found and / or expected to be found in a sample at a frequency lower than a value (e.g., a threshold) of a non-cancer or healthy (e.g., non-cancer) sample. Thus, for example, as used herein, the terms "abnormal methylation" and "anomalous methylation" describe nucleic acid (e.g., DNA, such as cfDNA) molecules or methylation state carriers that exhibit an abnormal methylation pattern. One aspect of differential methylation according to the present disclosure can include an aspect of abnormal methylation in some versions. Additionally, whether an aspect is differentially methylated can be used as an indicator of the health (e.g., not having cancer) of the subject from whom a sample of a subject is obtained, as opposed to the subject being diseased (e.g., having cancer). In some versions, the subject method includes determining whether a nucleic acid (e.g., DNA, molecule, or methylation state carrier) is abnormally methylated.

[0047] As used herein, the term "methylation state carrier" refers to a carrier that includes a plurality of elements, where each element indicates the methylation state of methylation sites in a nucleic acid (e.g., DNA) molecule that includes a plurality of methylation sites in the order in which the methylation sites occur in the DNA molecule from 5' to 3'. For example, <M x , M x+1 , M x+2 >, <M x , M x+1 , U x+2 >,... <Ux, U x+1 , U x+2 > can be a methylation carrier of a DNA molecule that includes three methylation sites, where M represents a methylated methylation site and U represents an unmethylated methylation site.

[0048] The terms "converted DNA molecule" and "converted cfDNA molecule" refer to these molecules obtained by processing DNA (e.g., cfDNA) molecules in a sample to distinguish methylated and unmethylated nucleotides in the DNA or cfDNA molecules. For example, in one embodiment, the sample can be subjected to bisulfite conversion and thus treated with bisulfite ions (e.g., using sodium bisulfite) to convert unmethylated cytosine ("C") to uracil ("U"). In another embodiment, the conversion of unmethylated cytosine to uracil is achieved by enzymatic conversion using an enzymatic conversion reaction (e.g., a reaction using a cytidine deaminase such as APOBEC). After treatment, the converted DNA molecule or cfDNA molecule includes additional uracils that were not present in the original cfDNA sample. Replication of the DNA strand containing uracil using DNA polymerase causes adenine to be added to the nascent complementary strand instead of guanine, which is normally added as a complement to cytosine or methylcytosine. In some embodiments, the converted DNA molecule is a converted hypermethylated DNA molecule.

[0049] The term "converted DNA sequence" refers to the sequence of a converted DNA molecule.

[0050] As used herein, the term "source tissue" or "TOO" refers to the organ, group of organs, body region, and / or cell type from which a nucleic acid (e.g., cfDNA) is derived, where the nucleic acid is, for example, health- or disease-related (e.g., cancer-related) cfDNA. Identifying the source tissue and / or cell type of a disease (e.g., cancer) can allow identification of the most appropriate subsequent steps in the disease care continuum for further diagnosis, staging, and treatment decisions.

[0051] Identifying Cell Types Based on DNA Methylation Status

[0052] The present disclosure provides compositions and methods for determining cell type based on the methylation status of associated DNA fragments. Such DNA fragments typically contain multiple adjacent CpG dinucleotides that have a relatively consistent methylation status (methylated or unmethylated) in one cell type. At the same time, the methylation status of such CpG sites is different in other cells, allowing the corresponding cell type to be distinguished from other cell types. Each individual CpG dinucleotide is referred to herein as a "CpG site". Similarly, a collection of multiple CpG sites within a DNA fragment is referred to as a "CpG cluster".

[0053] Previously, DNA methylation analysis mainly used bulk tissues and measured the average methylation of the detected CpG sites, thus excluding the study of rare cell types that may differ in DNA methylation, such as tissue-resident immune cells, fibroblasts, or endothelial cells. Alternatively, the analysis of cultured cells is often limited by the non-physiological methylation patterns introduced in vitro.

[0054] To overcome these limitations and accurately characterize the complexity of the human cell methylome, the inventors isolated FACS-purified populations of 39 primary human cell types from freshly dissociated adult healthy tissues. Different from many previous studies that used shallow sequencing or were limited to subsets of genomic regions (simplified representation as reduced representation bisulfite sequencing, RRBS), the present disclosure uses deep whole-genome sequencing in purified human cell populations, which has paired-end reads at an average sequencing depth of 32x (±7.2x). For each cell type, the analysis targets multiple replicates obtained from different individuals. The analysis combines read-specific methylation patterns across the entire genome into larger blocks, allowing simultaneous readout of the methylation status of multiple CpG sites that capture the dependencies between adjacent CpG sites, while reflecting the differences in methylation patterns across individual cell types.

[0055] As demonstrated in the attached experimental examples, surprisingly, in each of the large number of human cell types examined, a sufficient number of CpG clusters can be identified that have statistically different methylation states between one cell type and all other cell types. Such CpG clusters (also referred to as "methylation markers") allow the identification of each cell type based on its DNA methylation status.

[0056] According to one embodiment of the present disclosure, a method for identifying the cell type of DNA in a biological sample is provided. In some embodiments, the method requires detecting the methylation status of multiple CpG sites in a DNA fragment and then identifying the corresponding cell type based on the methylation status of these sites. According to various embodiments, the subject DNA fragment is derived from one or more cells of the determined cell type.

[0057] Methylation Detection

[0058] Detecting DNA methylation according to the subject embodiments can be implemented by various methods. In some embodiments, methylation is the conversion of cytosine to 5-methylcytosine (5-mC). In some embodiments, methylation is the conversion of cytosine to 5-hydroxymethylcytosine (5-hmC).

[0059] In some embodiments, the methylation status is detected directly, such as by mass spectrometry or methylation-sensitive restriction enzymes. A step of a DNA methylation method can produce a transformed DNA molecule. In such embodiments, methylated cytosines are transformed prior to further analysis. The terms "transformed" and "modified" refer to the treatment of DNA molecules in a sample to distinguish methylated nucleotides from unmethylated nucleotides. For example, in one embodiment, the sample can be treated with bisulfite ions (e.g., using sodium bisulfite) to convert unmethylated cytosine ("C") to uracil ("U"). In another embodiment, the conversion of unmethylated cytosine to uracil is achieved using an enzymatic conversion reaction (e.g., using cytidine deaminase, such as APOBEC-Seq (NEBiolabs, Ipswich, MA)). Examples of DNA methylation detection methods are further described below.

[0060] Methylation-specific PCR (MSP) can be based on the chemical reaction of bisulfite with DNA to convert unmethylated cytosine of CpG dinucleotides to uracil or UpG, followed by conventional PCR. Methylated cytosine will not be transformed during this process, and primers are designed to overlap with the CpG sites of interest, which allows technicians to determine the methylation status as methylated or unmethylated.

[0061] Whole-genome bisulfite sequencing (also known as BS-Seq) is a high-throughput whole-genome analysis of DNA methylation. This sequencing can also be based on the bisulfite conversion of genomic DNA, followed by sequencing on a next-generation sequencing platform, such as deep sequencing. The obtained sequences are then realigned with a reference genome to determine the methylation status of CpG dinucleotides based on the mismatches caused by the conversion of unmethylated cytosine to uracil.

[0062] HpaII tiny fragment enrichment by ligation-mediated PCR assay (HELP assay) compares the representations generated by digesting genomic DNA with restriction enzymes (e.g., HpaII or MspI), followed by ligation-mediated PCR. The HpaII representation enriches the hypomethylated fraction of the genome at the 5'-CCGG-3' sites where the cytosine in the central CG dinucleotide is unmethylated when digested by HpaII.

[0063] Glal hydrolysis and ligation adaptor-dependent PCR assay (GLAD-PCR assay) can identify R(5mC)GY sites generated during de novo DNA methylation by DNMT 3A and DNMT 3B DNA methyltransferases. The GLAD-PCR assay does not require bisulfite treatment of DNA. The GLAD-PCR assay uses a site-specific methylation-directed DNA endonuclease (MD DNA endonuclease) that cleaves only methylated DNA and not unmethylated DNA.

[0064] The "Illumina methylation assay" uses array hybridization to measure locus-specific DNA methylation. Bisulfite-treated DNA is hybridized to probes on a "BeadChip". Single-base extension of labeled probes is used to determine the methylation status of target sites. The Infinium MethylationEPIC BeadChip can interrogate over 850,000 methylation sites across the entire human genome.

[0065] The "Enzymatic Methyl-seq" or "EM-seq" method developed by New England Biolabs provides an alternative to bisulfite modification. This method relies on the ability of APOBEC (e.g., APOBEC-Seq developed by NEB) to deaminate cytosine to uracil. Then, cytosine is considered thymine in sequencing, and methylated cytosine is considered cytosine in sequencing.

[0066] DNA Sample Preparation

[0067] DNA fragments subjected to methylation status detection can be prepared from cell-containing or cell-free samples. Cell-containing biological samples can be readily obtained, such as from biopsies, cultured cells, skin tissue, cells, body fluids, but are not limited thereto. In some embodiments, the cell-containing biological sample is tumor tissue or tumor cells. In some embodiments, the cell-containing biological sample is a body fluid sample containing at least one cell. Non-limiting examples of body fluids that can be used according to the subject method include blood, plasma, serum, semen, milk, urine, vaginal secretions, uterine or vaginal lavage fluids, pleural effusions, ascites, sweat, tears, sputum, bronchoalveolar lavage fluid, feces, saliva, and cerebrospinal fluid.

[0068] In some embodiments, cell-free DNA samples can also be used. Cell-free DNA circulates in an individual's body and can be derived from healthy cells, or diseased, aging, or damaged cells. For pregnant women, cell-free DNA can also be derived from the fetus. In some embodiments, cell-free DNA is obtained from a biological sample that includes blood, plasma, serum, semen, milk, urine, saliva, or cerebrospinal fluid, or any other body fluid or tissue.

[0069] DNA fragments can be isolated from biological samples by methods known in the art. In some embodiments, the DNA fragments are substantially free of proteins, lipids, and other common substances from tissue or fluid samples. In some embodiments, the length of the DNA fragments is suitable for methylation analysis.

[0070] In some embodiments, the average length of the DNA fragments is at least 18 bp, 20 bp, 25 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 110 bp, 120 bp, 130 bp, 140 bp, 150 bp, 200 bp, 250 bp, 300 bp, or 350 bp. In some embodiments, the average length of the DNA fragments does not exceed 100 bp, 110 bp, 120 bp, 130 bp, 140 bp, 150 bp, 160 bp, 170 bp, 180 bp, 190 bp, 200 bp, 210 bp, 220 bp, 230 bp, 240 bp, 250 bp, 260 bp, 270 bp, 280 bp, 290 bp, 300 bp, or 350 bp. In some embodiments, the average length of the DNA fragments is from 40 bp to 300 bp, 400 bp to 250 bp, 40 bp to 200 bp, 50 bp to 300 bp, 50 bp to 250 bp, 50 bp to 200 bp, 50 bp to 150 bp, 100 bp to 300 bp, 100 bp to 250 bp, 100 bp to 200 bp, or 150 bp to 300 bp, but is not limited thereto.

[0071] In some embodiments, the DNA fragments from the biological sample are processed to obtain a desired average length. This can be achieved, for example, by sonication. In some embodiments, the desired average length can be obtained by enriching DNA fragments of the desired length while discarding fragments that are too short or too long, such as by liquid chromatography.

[0072] In some embodiments, even if the average length of the DNA fragments is longer than the desired length, it is not necessary to degrade these DNA fragments. Alternatively, DNA methylation detection can be limited to the desired fragment / sequence, which is achieved by designing appropriate primers (e.g., in methylation-specific PCR) or by targeted mapping of the methylation status detected within the desired fragment / sequence.

[0073] Methylation detection can be performed on the prepared DNA fragments. In some embodiments, it is desirable to detect the methylation status of CpG sites that are adjacent to each other and together form a CpG cluster. As used herein, the term "adjacent" refers to CpG sites that are all located within a certain region on a DNA fragment. In some embodiments, the length of this region does not exceed 100bp, 110bp, 120bp, 130bp, 140bp, 150bp, 160bp, 170bp, 180bp, 190bp, 200bp, 210bp, 220bp, 230bp, 240bp, 250bp, 260bp, 270bp, 280bp, 290bp, 300bp, 350bp, 400bp, 450bp or 500bp. In some embodiments, two CpG sites are considered adjacent when the distance between one CpG site and another CpG site does not exceed 100bp, 110bp, 120bp, 130bp, 140bp, 150bp, 160bp, 170bp, 180bp, 190bp, 200bp, 210bp, 220bp, 230bp, 240bp, 250bp, 260bp, 270bp, 280bp, 290bp, 300bp, 350bp, 400bp, 450bp or 500bp.

[0074] In some embodiments, the methylation status of at least three adjacent CpG sites is detected. In some embodiments, the methylation status of at least four adjacent CpG sites is detected. In some embodiments, the methylation status of at least five adjacent CpG sites is detected. In some embodiments, the methylation status of at least six adjacent CpG sites is detected. In some embodiments, the methylation status of at least seven adjacent CpG sites is detected. In some embodiments, the methylation status of at least eight adjacent CpG sites is detected. In some embodiments, the methylation status of at least nine adjacent CpG sites is detected. In some embodiments, the methylation status of at least ten adjacent CpG sites is detected. In some embodiments, the methylation status of at least 11, 12, 13, 14, or 15 adjacent CpG sites is detected. In some embodiments, the methylation status of at least three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen CpG sites is detected. Each of such sites may be completely non - adjacent or partially non - adjacent to other sites. For example, one site may be adjacent to another site on one side and non - adjacent on the opposite side, or may be non - adjacent to other sites on both sides.

[0075] Use of Methylation Markers

[0076] The methylation status of these adjacent CpG sites on a DNA fragment can be used, according to the subject method, to identify the cell type of the source cell of the DNA fragment. In some embodiments, the methylation status of these CpG sites is the frequency of methylated CpG sites, which can be expressed as a percentage (M%). For example, for a DNA fragment F1 of length 200 bp and including 10 CpG sites, when two of these CpG sites are methylated and eight are unmethylated, the methylation status of the DNA fragment in natural killer cells can be expressed as 20%. If the average methylation status of F1 in all other cell types (i.e., cell types other than natural killer cells) is in the range of 70% to 90%, then F1 can be a suitable marker for identifying natural killer cells. For example, according to the subject method, it can be determined that cell - free DNA is released from natural killer cells, and the cell - free DNA includes F1, where two of the 10 CpG sites within F1 are methylated.

[0077] In some embodiments, truncated methylation percentage values can be used when determining cell types. Such truncated values can be determined based on experimental data, such as those presented in the appended experimental examples, which have appropriate statistics and are applied according to the subject methods. For example, if the methylation percentage of F1 in all tested natural killer cells is in the range of 0 to 40%, and the methylation percentage of F1 in all tested non-natural killer cells is in the range of 60% to 100%, then 50% can be used as an appropriate truncated value. It should be understood that a truncated value is not always required. For example, when the methylation status of an F1 fragment from an unknown cell is detected and shows 30% methylation, this 30% number can be compared with F1 from natural killer cells and non-natural killer cells, and the nearest neighbors can be analyzed and applied to determine the type of the unknown cell.

[0078] In some embodiments, the methylation status of multiple DNA fragments can be used together in a multivariate analysis manner to determine the type of a cell. For example, when analyzing cancer cells of unknown primary origin, the methylation status of DNA fragments F1, F2, and F3 can be detected. Methods such as random forest, linear regression, support vector machine, and nearest neighbor method (but not limited to these) can be used to determine the primary cell type of the cancer cells using multiple methylation percentages.

[0079] Disease Detection and Treatment Monitoring

[0080] Cell type identification has important clinical uses. For example, in many diseases, DNA from dying cells is released into the bloodstream or other body fluids (e.g., semen, milk, urine, saliva, and cerebrospinal fluid). Tools that can identify the source tissue of this DNA can be used to identify and localize diseases. Also, changes in the amount of this released DNA can indicate disease progression or treatment efficacy. For example, the subject methods include measuring the amount of this released DNA at multiple time points (such as a first time point and a second time point later than the first time point). In some versions, measurements are also made at a third time point after the second time point and / or at consecutive time points after the second time point. In some versions, the second time point or additional such time points are after a disease (e.g., cancer) treatment is administered to a subject (e.g., after resection surgery and / or therapeutic intervention), and / or the first time point is before such treatment. These methods can include determining whether a disease (e.g., cancer) is worsening or improving based on the difference in the amount of DNA between more than two (e.g., more than 3, more than 4, more than 5, or more than 10) time points. For example, an increase in the amount of disease (e.g., cancer) DNA can indicate that the disease (e.g., cancer) condition is worsening, while a decrease in this DNA amount can indicate that the disease condition is improving. Thus, the subject methods can include providing a disease diagnosis and / or treatment regimen based on the difference between the determined multiple measurements.

[0081] In addition, for cancers of unknown primary origin (CUP), identifying the cell type can help identify its primary origin, which may be crucial for providing an initial disease diagnosis and / or confirming an appropriate treatment regimen.

[0082] The subject methods can include detecting, such as detecting tissue originating from but not limited to the following: carcinoma, lymphoma, embryonal tumor, sarcoma, and leukemia or lymphoid malignancies. Specific examples of cancers can include but are not limited to: liver cancer (e.g., hepatocellular carcinoma (F1CC)), hepatoma, hepatic carcinoma, bladder cancer (e.g., urothelial bladder cancer), testicular (germ cell tumor) cancer, breast cancer (e.g., HER2-positive, HER2-negative, and triple-negative breast cancer), brain cancer (e.g., astrocytoma, glioma (e.g., glioblastoma)), colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer (e.g., renal cell carcinoma, nephroblastoma or Wilms' tumor), prostate cancer, vulvar cancer, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), skin cancer, melanoma, lung cancer (including small cell lung cancer, non-small cell lung cancer (“NSCLC”), lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, stomach cancer (including gastrointestinal cancer), pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), cervical cancer, ovarian cancer (e.g., high-grade serous ovarian cancer), thyroid cancer, anal cancer, penile cancer, head and neck cancer, esophageal cancer, and nasopharyngeal cancer (NPC). Additional examples of cancers include but are not limited to: fibrosarcoma, choriocarcinoma, laryngeal cancer, retinoblastoma, granulosa cell tumor, androblastoma, hematological malignancies, including but not limited to non-Hodgkin lymphoma (NHL), multiple myeloma, and acute hematological malignancies, endometriosis, Kaposi's sarcoma, rhabdomyosarcoma, osteosarcoma, leiomyosarcoma, urinary tract cancer, schwannoma, oligodendroglioma, and neuroblastoma.

[0083] In some embodiments, cancers according to the present disclosure can be uterine cancer, upper gastrointestinal squamous cell carcinoma, all other upper gastrointestinal cancers, thyroid cancer, sarcoma, urothelial renal carcinoma, all other renal carcinomas, prostate cancer, pancreatic cancer, ovarian cancer, neuroendocrine cancer, multiple myeloma, melanoma, lymphoma, small cell lung cancer, lung adenocarcinoma, all other lung cancers, leukemia, hepatobiliary tract cancer, hepatobiliary tract cholangiocarcinoma, head and neck cancer, colorectal cancer, cervical cancer, breast cancer, bladder cancer, anal rectal cancer, or any combination thereof. Cancers according to the subject embodiments can also be anal cancer, esophageal cancer, head and neck cancer, liver / bile duct cancer, lung cancer, ovarian cancer, pancreatic cancer, plasma cell tumor, gastric cancer, or any combination thereof. Cancers according to the subject embodiments can be thyroid cancer, melanoma, myeloid tumors, renal cancer, prostate cancer, breast cancer, uterine cancer, ovarian cancer, bladder cancer, urothelial cancer, cervical cancer, anal rectal cancer, head and neck cancer, colorectal cancer, liver cancer, bile duct cancer, pancreatic cancer, gallbladder cancer, upper gastrointestinal cancer, multiple myeloma, lymphoid tumors, lung cancer, or any combination thereof.

[0084] Regarding example cell types and groups of cell types, various examples of the clinical applications of the technology of the present invention are described in further detail below.

[0085] A. Gastrointestinal Cells

[0086] The gastrointestinal (GI) system, or the gastrointestinal tract, is a tube that runs from the mouth to the anus and includes all the organs of the digestive system in humans and other animals. Food ingested through the mouth is digested to extract nutrients and absorb energy, and then waste is excreted as feces. Given the shared functions of these organs, various different types of cells and tissues in this system share some common molecular characteristics, including genetic and epigenetic characteristics.

[0087] A.1. Oral, Laryngeal, and Esophageal Epithelial Cells

[0088] It has been found herein that some genomic locations in oral, laryngeal, and esophageal epithelial cells are consistently hypomethylated or hypermethylated compared to all other cell types in humans (see, for example, Table A). For example, genomic sequences provided as SEQ ID NO: 1-15, 16-90, 91-91, 92-101, or 102-125 (annotated with start and end positions on their respective chromosomes) all have a methylation percentage of less than 40% in oral, laryngeal, or esophageal epithelial cells, while having a methylation percentage of greater than 60% in all other cell types. Similarly, genomic sequences provided as SEQ ID NO: 126-133, 134-134, or 135-150 have a relatively high methylation percentage (>60%) in oral, laryngeal, or esophageal epithelial cells, while having a lower methylation percentage (<40%) in all other cell types.

[0089] Table A. List of Markers

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107] *U: Lower (unmethylated) methylation in specific cell types and higher methylation in other cell types;

[0108] M: Higher (methylated) methylation in specific cell types and lower methylation in other cell types.

[0109] Each genomic sequence in the Sequence Listing (according to the human genome version hg19, Genome Reference Consortium Human Build 37 (GRCh37), published on February 27, 2009) represents a DNA fragment that includes or overlaps with that genomic sequence. In some embodiments, the DNA fragment that includes a CpG cluster that can be used as a methylation marker includes at least the CpG sites contained in the genomic sequence as defined in the Sequence Listing. In some embodiments, the DNA fragment includes at least two, three, four, five, six, seven, eight, nine, ten, or more CpG sites contained in the genomic sequence as defined in the Sequence Listing.

[0110] The Sequence Listing is submitted in ASCII format, and all of its content is hereby incorporated by reference. A list of all sequences is provided in Table B, but no actual sequences are included. Each sequence (see the example shown in Table C) is annotated with its genomic location (e.g., chr9: 119238427-119238709), nearby genes, location (e.g., intron of ASTN2), and region, the corresponding cell type (e.g., oral, laryngeal, and esophageal epithelium), whether it is hypomethylated (U) or "hypermethylated (M)" in the corresponding cell type, and the average methylation frequency in that cell type compared to the average methylation frequency in all other cell types (e.g., 0.05: 0.94).

[0111] Table B. Target Sequences

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439]

[0440]

[0441]

[0442]

[0443]

[0444]

[0445]

[0446]

[0447]

[0448]

[0449]

[0450]

[0451]

[0452]

[0453]

[0454]

[0455]

[0456]

[0457]

[0458] Table C. Example Sequences and Annotations in the Sequence Listing

[0459]

[0460]

[0461] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from oral, laryngeal, or esophageal epithelial cells is provided. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more) CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten, or all) of the CpG sites is located within a human genomic sequence selected from SEQ ID NOs: 1-15 or 16-90, or is less than 100 bp, 200 bp, 500 bp, or 1 kb away from the foregoing human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from oral, laryngeal, or esophageal epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from oral, laryngeal, or esophageal epithelial cells. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from oral, laryngeal, or esophageal epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from oral, laryngeal, or esophageal epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from oral, laryngeal, or esophageal epithelial cells.

[0462] In some embodiments, the method requires detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites in a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites are within a human genomic sequence selected from SEQ ID NO: 126 - 133, or are within less than 100 bp, 200 bp, 500 bp or 1 kb of the foregoing human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from oral, laryngeal or esophageal epithelial cells. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from oral, laryngeal or esophageal epithelial cells. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from oral, laryngeal or esophageal epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from oral, laryngeal or esophageal epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from oral, laryngeal or esophageal epithelial cells.

[0463] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by genomic sequences SEQ ID NO: 1 - 15, 16 - 90, 91 - 91, 92 - 101, 102 - 125, 126 - 133, 134 - 134 or 135 - 150.

[0464] In some embodiments, the method requires detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites in a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites are located within a human genomic sequence selected from SEQ ID NO: 1-15, 16-90, 91-91, 92-101 or 102-125, or are within less than 100 bp, 200 bp, 500 bp or 1 kb of the foregoing human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from oral, laryngeal or esophageal epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from oral, laryngeal or esophageal epithelial cells. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from oral, laryngeal or esophageal epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from oral, laryngeal or esophageal epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from oral, laryngeal or esophageal epithelial cells.

[0465] In some embodiments, the method requires detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) CpG sites are located within a human genomic sequence selected from SEQ ID NO: 126 - 133, 134 - 134 or 135 - 150, or are less than 100 bp, 200 bp, 500 bp or 1 kb away from the foregoing human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method subsequently identifies that the target DNA fragment is from oral, laryngeal or esophageal epithelial cells. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from oral, laryngeal or esophageal epithelial cells. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from oral, laryngeal or esophageal epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from oral, laryngeal or esophageal epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from oral, laryngeal or esophageal epithelial cells.

[0466] In some embodiments, when the prediction results according to two or more of the above methods are consistent with each other, the prediction results are further confirmed.

[0467] The cell type identification method can be used to detect diseases or conditions associated with cell types. In one embodiment, when cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva or cerebrospinal fluid) is identified as being from oral, laryngeal or esophageal epithelial cells of a subject, the method indicates that the subject has abnormal cell death and / or suffers from a disease associated with that cell. In some embodiments, the disease or condition is an injury, inflammation or cancer of the oral, laryngeal or esophageal epithelium.

[0468] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., oral, laryngeal, or esophageal epithelial cells) decreases (e.g., less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., oral, laryngeal, or esophageal epithelial cells) increases (e.g., more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two detections, the subject undergoes treatment, so the detection results indicate the treatment effect.

[0469] In one embodiment, a method for determining the cell type of disease cells (e.g., cancer cells), the primary origin of the disease (e.g., cancer cells), or the signal or source of the disease (e.g., cancer cells) is also provided. In some embodiments, the primary origin of the cancer cells is unknown. In some embodiments, the methods described above include detecting the methylation status of one or more DNA fragments of the cancer cells, and the methylation status can then be used to determine that the cells are oral, laryngeal, or esophageal epithelial cells, as described above.

[0470] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of cancer cells. In some embodiments, genetic variations can also be present in the DNA fragments, so cell type detection can help associate the genetic variations with cancer. In some embodiments, the genetic variations include mutations. In some embodiments, the genetic variations include deletions or insertions. In some embodiments, the genetic variations constitute microsatellite instability. In some embodiments, the genetic variations constitute loss of heterozygosity. In some embodiments, the genetic variations disrupt or alter gene splicing. In some embodiments, the genetic variations cause frameshifts or generate premature stop codons.

[0471] Once the primary origin of the cancer is identified, the subject can be treated with an appropriate regimen for that cancer type.

[0472] A2. Gastric Epithelium

[0473] As also provided in Table A, some genomic locations in gastric epithelial cells are consistently hypomethylated or hypermethylated compared to all other cell types in humans.

[0474] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from gastric epithelial cells is provided. In some embodiments, the method requires detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites in a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites is located within a human genomic sequence selected from SEQ ID NO: 151-170, 171-330, 331-335, 336-340 or 341-378 or selected from SEQ ID NO: 151-170 or 171-330, or is less than 100 bp, 200 bp, 500 bp or 1 kb away from the aforementioned human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from gastric epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from gastric epithelial cells. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from gastric epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from gastric epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from gastric epithelial cells.

[0475] In some embodiments, the method requires detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) CpG sites are located within a human genomic sequence selected from SEQ ID NO: 379-401, 402-402 or 403-428 or within a human genomic sequence selected from SEQ ID NO: 379-401, or are less than 100 bp, 200 bp, 500 bp or 1 kb away from the aforementioned human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from gastric epithelial cells. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from gastric epithelial cells. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from gastric epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from gastric epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from gastric epithelial cells.

[0476] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine the cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by the genomic sequences SEQ ID NO: 151-170, 171-330, 331-335, 336-340, 341-378, 379-401, 402-402 or 403-428.

[0477] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, when the cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva or cerebrospinal fluid) is identified as being from the gastric epithelial cells of a subject, the method indicates that the subject has abnormal cell death and / or suffers from a disease associated with the cell. In some embodiments, the disease or condition is damage, inflammation or cancer of the gastric epithelium.

[0478] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., gastric epithelial cells) decreases (e.g., less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., gastric epithelial cells) increases (e.g., more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two detections, the subject undergoes treatment, and thus the detection results indicate the treatment effect.

[0479] In one embodiment, a method for determining the cell type of disease cells (e.g., cancer cells), the primary origin of the disease (e.g., cancer cells), or the signal or origin of the disease (e.g., cancer cells) is also provided. In some embodiments, the primary origin of the cancer cells is unknown. In some embodiments, the methods described above include detecting the methylation status of one or more DNA fragments of the cancer cells, and the methylation status can then be used to determine that the cells are gastric epithelial cells, as described above.

[0480] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of the cancer cells. In some embodiments, genetic variations can also be present in the DNA fragments, and thus cell type detection can help associate the genetic variations with cancer. In some embodiments, the genetic variations include mutations. In some embodiments, the genetic variations include deletions or insertions. In some embodiments, the genetic variations constitute microsatellite instability. In some embodiments, the genetic variations constitute loss of heterozygosity. In some embodiments, the genetic variations disrupt or alter gene splicing. In some embodiments, the genetic variations cause a frameshift or generate a premature stop codon. Once the primary origin of the cancer is identified, the subject can be treated with an appropriate regimen for that cancer type.

[0481] A3. Small Intestinal Epithelium

[0482] Also as provided in Table A, some genomic locations in small intestinal epithelial cells are consistently hypomethylated or hypermethylated compared to all other cell types in humans.

[0483] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from small intestinal epithelial cells is provided. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites are located within a human genomic sequence selected from SEQ ID NO: 429 - 446, 447 - 527, 528 - 529, 530 - 536 or 537 - 554 or selected from SEQ ID NO: 429 - 446 or 447 - 527, or are within less than 100 bp, 200 bp, 500 bp or 1 kb of the foregoing human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from small intestinal epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from small intestinal epithelial cells. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from small intestinal epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from small intestinal epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from small intestinal epithelial cells.

[0484] In some embodiments, the method requires detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) CpG sites are located within a human genomic sequence selected from SEQ ID NO: 555 - 564, 565 - 565 or 566 - 579 or within a human genomic sequence selected from SEQ ID NO: 555 - 564, or are within less than 100 bp, 200 bp, 500 bp or 1 kb of the foregoing human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from small intestinal epithelial cells. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from small intestinal epithelial cells. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from small intestinal epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from small intestinal epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from small intestinal epithelial cells.

[0485] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by the genomic sequences SEQ ID NO: 429 - 446, 447 - 527, 528 - 529, 530 - 536, 537 - 554, 555 - 564, 565 - 565 or 566 - 579.

[0486] The cell type identification method can be used to detect diseases or conditions associated with cell type. In one embodiment, when cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva or cerebrospinal fluid) is identified as being from the small intestinal epithelial cells of a subject, the method indicates that the subject has abnormal cell death and / or has a disease associated with that cell. In some embodiments, the disease or condition is injury, inflammation or cancer of the small intestinal epithelium.

[0487] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., small intestinal epithelial cells) decreases (e.g., is less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., small intestinal epithelial cells) increases (e.g., is more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two tests, the subject undergoes treatment, so the test results indicate the treatment effect.

[0488] In one embodiment, a method for determining the cell type of disease cells (e.g., cancer cells), the primary origin of the disease (e.g., cancer cells), or the signal or origin of the disease (e.g., cancer cells) is also provided. In some embodiments, the primary origin of the cancer cells is unknown. In some embodiments, the methods described above include detecting the methylation status of one or more DNA fragments of the cancer cells, and the methylation status can then be used to determine that the cells are small intestinal epithelial cells, as described above.

[0489] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, so cell type detection can help associate the genetic variations with cancer. In some embodiments, the genetic variations include mutations. In some embodiments, the genetic variations include deletions or insertions. In some embodiments, the genetic variations constitute microsatellite instability. In some embodiments, the genetic variations constitute loss of heterozygosity. In some embodiments, the genetic variations disrupt or alter gene splicing. In some embodiments, the genetic variations cause frameshifts or generate premature stop codons. Once the primary origin of the cancer is identified, the subject can be treated with an appropriate regimen for that cancer type.

[0490] A4. Colonic Epithelium

[0491] Also as provided in Table A, some genomic locations in colon epithelial cells are consistently undermethylated or overmethylated compared to all other cell types in humans.

[0492] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from colonic epithelial cells is provided. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites is located within a human genomic sequence selected from SEQ ID NO: 580-596, 597-657, 658-660, 661-668 or 669-704 or selected from SEQ ID NO: 580-596 or 597-657, or is less than 100 bp, 200 bp, 500 bp or 1 kb away from the aforementioned human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from colonic epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from colonic epithelial cells. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from colonic epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from colonic epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from colonic epithelial cells.

[0493] In some embodiments, the method requires detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) CpG sites are located within a human genomic sequence selected from SEQ ID NO: 705 - 715 or 716 - 729 or within a human genomic sequence selected from SEQ ID NO: 705 - 715, or within 100 bp, 200 bp, 500 bp or 1 kb of the foregoing human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from a colonic epithelial cell. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from a colonic epithelial cell. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from a colonic epithelial cell. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from a colonic epithelial cell. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from a colonic epithelial cell.

[0494] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine the cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by the genomic sequences SEQ ID NO: 580 - 596, 597 - 657, 658 - 660, 661 - 668, 669 - 704, 705 - 715 or 716 - 729.

[0495] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, when the cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva or cerebrospinal fluid) is identified as being from a subject's colonic epithelial cells, the method indicates that the subject has abnormal cell death and / or has a disease associated with the cell. In some embodiments, the disease or condition is damage, inflammation or cancer of the colonic epithelial cells.

[0496] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., colon epithelial cells) decreases (e.g., is less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., colon epithelial cells) increases (e.g., is more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two tests, the subject undergoes treatment, so the test results indicate the treatment effect.

[0497] In one embodiment, a method is also provided for determining the cell type of disease cells (e.g., cancer cells), the primary origin of the disease (e.g., cancer cells), or the signal or origin of the disease (e.g., cancer cells). In some embodiments, the primary origin of the cancer cells is unknown. In some embodiments, the methods described above include detecting the methylation status of one or more DNA fragments of the cancer cells, and the methylation status can then be used to determine that the cells are colon epithelial cells, as described above.

[0498] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of the cancer cells. In some embodiments, genetic variations can also be present in the DNA fragments, so cell type detection can help associate the genetic variations with cancer. In some embodiments, the genetic variations include mutations. In some embodiments, the genetic variations include deletions or insertions. In some embodiments, the genetic variations constitute microsatellite instability. In some embodiments, the genetic variations constitute loss of heterozygosity. In some embodiments, the genetic variations disrupt or alter gene splicing. In some embodiments, the genetic variations cause a frameshift or generate a premature stop codon. Once the primary origin of the cancer is identified, the subject can be treated with an appropriate regimen for that cancer type.

[0499] A5. Colonic Fibroblasts

[0500] Also as provided in Table A, some genomic locations in colon fibroblasts are consistently undermethylated or overmethylated compared to all other cell types in humans.

[0501] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from colonic fibroblasts is provided. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites are located within a human genomic sequence selected from SEQ ID NO: 730 - 732, or are less than 100 bp, 200 bp, 500 bp or 1 kb away from the aforementioned human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from colonic fibroblasts. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from colonic fibroblasts. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from colonic fibroblasts. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from colonic fibroblasts. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from colonic fibroblasts.

[0502] In some embodiments, the method requires detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) CpG sites are located within a human genomic sequence selected from SEQ ID NO: 733-739 or 740-741 or selected from SEQ ID NO: 733-739, or are less than 100 bp, 200 bp, 500 bp or 1 kb away from the foregoing human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from a colon fibroblast. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from a colon fibroblast. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from a colon fibroblast. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from a colon fibroblast. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from a colon fibroblast.

[0503] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine the cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by the genomic sequences SEQ ID NO: 730-732, 733-739 or 740-741.

[0504] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, when cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva or cerebrospinal fluid) is identified as being from a colon fibroblast of a subject, the method indicates that the subject has abnormal cell death and / or has a disease associated with the cell. In some embodiments, the disease or condition is damage, inflammation or cancer of the colon fibroblast.

[0505] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., colonic fibroblasts) decreases (e.g., less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., colonic fibroblasts) increases (e.g., more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two detections, the subject undergoes treatment, so the detection results indicate the treatment effect.

[0506] In one embodiment, a method for determining the cell type of disease cells (e.g., cancer cells), the primary origin of the disease (e.g., cancer cells), or the signal or origin of the disease (e.g., cancer cells) is also provided. In some embodiments, the primary origin of the cancer cells is unknown. In some embodiments, the methods described above include detecting the methylation status of one or more DNA fragments of the cancer cells, and the methylation status can then be used to determine that the cells are colonic fibroblasts, as described above.

[0507] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of cancer cells. In some embodiments, genetic variations can also be present in the DNA fragments, so cell type detection can help associate the genetic variations with cancer. In some embodiments, the genetic variations include mutations. In some embodiments, the genetic variations include deletions or insertions. In some embodiments, the genetic variations constitute microsatellite instability. In some embodiments, the genetic variations constitute loss of heterozygosity. In some embodiments, the genetic variations disrupt or alter gene splicing. In some embodiments, the genetic variations cause frameshifts or generate premature stop codons. Once the primary origin of the cancer is identified, the subject can be treated with an appropriate regimen for that cancer type.

[0508] A6. Gallbladder Epithelium

[0509] Also as provided in Table A, some genomic locations in gallbladder epithelial cells are consistently hypomethylated or hypermethylated compared to all other cell types in humans.

[0510] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from gallbladder epithelial cells is provided. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites are located within a human genomic sequence selected from SEQ ID NO: 742-758, 759-829, 830-831, 832-839 or 840-867 or selected from SEQ ID NO: 742-758 or 759-829, or are less than 100 bp, 200 bp, 500 bp or 1 kb away from the aforementioned human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from gallbladder epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from gallbladder epithelial cells. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from gallbladder epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from gallbladder epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from gallbladder epithelial cells.

[0511] In some embodiments, the method requires detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) CpG sites are located within a human genomic sequence selected from SEQ ID NO: 868-875 or 876-876 or within a human genomic sequence selected from SEQ ID NO: 868-875, or are less than 100 bp, 200 bp, 500 bp or 1 kb away from the foregoing human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from gallbladder epithelial cells. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from gallbladder epithelial cells. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from gallbladder epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from gallbladder epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from gallbladder epithelial cells.

[0512] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by the genomic sequences SEQ ID NO: 742-758, 759-829, 830-831, 832-839, 840-867, 868-875 or 876-876.

[0513] The cell type identification method can be used to detect diseases or conditions associated with cell type. In one embodiment, when cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva or cerebrospinal fluid) is identified as being from the gallbladder epithelial cells of a subject, the method indicates that the subject has abnormal cell death and / or has a disease associated with the cell. In some embodiments, the disease or condition is damage, inflammation or cancer of the gallbladder epithelium.

[0514] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., gallbladder epithelial cells) decreases (e.g., is less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., gallbladder epithelial cells) increases (e.g., is more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two tests, the subject undergoes treatment, so the test results indicate the treatment effect.

[0515] In one embodiment, a method is also provided for determining the cell type of disease cells (e.g., cancer cells), the primary origin of the disease (e.g., cancer cells), or the signal or source of the disease (e.g., cancer cells). In some embodiments, the primary origin of the cancer cells is unknown. In some embodiments, the methods described above include detecting the methylation status of one or more DNA fragments of the cancer cells, and the methylation status can then be used to determine that the cells are gallbladder epithelial cells, as described above.

[0516] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of the cancer cells. In some embodiments, genetic variations can also be present in the DNA fragments, so cell type detection can help associate the genetic variations with cancer. In some embodiments, the genetic variations include mutations. In some embodiments, the genetic variations include deletions or insertions. In some embodiments, the genetic variations constitute microsatellite instability. In some embodiments, the genetic variations constitute loss of heterozygosity. In some embodiments, the genetic variations disrupt or alter gene splicing. In some embodiments, the genetic variations cause a frameshift or generate a premature stop codon. Once the primary origin of the cancer is identified, the subject can be treated with an appropriate regimen for that cancer type.

[0517] A7. Hepatocytes of the Liver

[0518] Also as provided in Table A, some genomic locations in liver hepatocytes are consistently hypomethylated or hypermethylated compared to all other cell types in humans.

[0519] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from liver hepatocytes is provided. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites is located within a human genomic sequence selected from SEQ ID NO: 877-896, 897-980, 981-983, 984-986, 987-988 or 989-1002 or selected from SEQ ID NO: 877-896 or 897-980, or is less than 100 bp, 200 bp, 500 bp or 1 kb away from the aforementioned human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from liver hepatocytes. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from liver hepatocytes. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from liver hepatocytes. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from liver hepatocytes. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from liver hepatocytes.

[0520] In some embodiments, the method requires detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) CpG sites are located within a human genomic sequence selected from SEQ ID NO: 1003-1018, 1019-1023 or 1024-1027 or selected from SEQ ID NO: 1003-1018, or are less than 100 bp, 200 bp, 500 bp or 1 kb away from the foregoing human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method subsequently identifies that the target DNA fragment is from liver hepatocytes. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from liver hepatocytes. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from liver hepatocytes. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from liver hepatocytes. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from liver hepatocytes.

[0521] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine the cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by the genomic sequences SEQ ID NO: 877-896, 897-980, 981-983, 984-986, 987-988, 989-1002, 1003-1018, 1019-1023 or 1024-1027.

[0522] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, when the cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva or cerebrospinal fluid) is identified as being from the liver hepatocytes of a subject, the method indicates that the subject has abnormal cell death and / or suffers from a disease associated with the cell. In some embodiments, the disease or condition is damage, inflammation or cancer of the liver hepatocytes.

[0523] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., liver hepatocytes) decreases (e.g., is less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition based on the recovery indication accordingly. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., liver hepatocytes) increases (e.g., is more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition based on the worsening indication accordingly. In some embodiments, between more than two tests, the subject undergoes treatment, so the test results indicate the treatment effect.

[0524] In one embodiment, a method for determining the cell type of a disease cell (e.g., a cancer cell), the primary source of a disease (e.g., a cancer cell), or the signal or origin of a disease (e.g., a cancer cell) is also provided. In some embodiments, the primary source of the cancer cell is unknown. In some embodiments, the methods described above include detecting the methylation status of one or more DNA fragments of the cancer cell, and the methylation status can then be used to determine that the cell is a liver hepatocyte, as described above.

[0525] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, so cell type detection can help associate the genetic variations with cancer. In some embodiments, the genetic variations include mutations. In some embodiments, the genetic variations include deletions or insertions. In some embodiments, the genetic variations constitute microsatellite instability. In some embodiments, the genetic variations constitute loss of heterozygosity. In some embodiments, the genetic variations disrupt or alter gene splicing. In some embodiments, the genetic variations cause a frameshift or generate a premature stop codon. Once the primary source of the cancer is identified, the subject can be treated with an appropriate regimen for that cancer type.

[0526] A8. Pancreatic Acinar Cells

[0527] Also as provided in Table A, some genomic locations in pancreatic acinar cells are consistently hypomethylated or hypermethylated compared to all other cell types in humans.

[0528] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from pancreatic acinar cells is provided. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites in a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites is located within a human genomic sequence selected from SEQ ID NO: 1028 - 1041, 1042 - 1112, 1113 - 1116, 1117 - 1127 or 1128 - 1155 or selected from SEQ ID NO: 1028 - 1041 or 1042 - 1112, or is less than 100 bp, 200 bp, 500 bp or 1 kb away from the foregoing human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from pancreatic acinar cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from pancreatic acinar cells. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from pancreatic acinar cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from pancreatic acinar cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from pancreatic acinar cells.

[0529] In some embodiments, the method requires detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) CpG sites are located within a human genomic sequence selected from SEQ ID NOs: 1156 - 1161 or 1162 - 1180 or within a human genomic sequence selected from SEQ ID NOs: 1156 - 1161, or are within less than 100 bp, 200 bp, 500 bp or 1 kb of the foregoing human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from pancreatic acinar cells. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from pancreatic acinar cells. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from pancreatic acinar cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from pancreatic acinar cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from pancreatic acinar cells.

[0530] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by the genomic sequences SEQ ID NOs: 1028 - 1041, 1042 - 1112, 1113 - 1116, 1117 - 1127, 1128 - 1155, 1156 - 1161 or 1162 - 1180.

[0531] The cell type identification method can be used to detect diseases or conditions associated with cell type. In one embodiment, when cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva or cerebrospinal fluid) is identified as being from pancreatic acinar cells of a subject, the method indicates that the subject has abnormal cell death and / or has a disease associated with that cell. In some embodiments, the disease or condition is injury, inflammation or cancer of pancreatic acinar cells. In some embodiments, the disease is diabetes.

[0532] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., pancreatic acinar cells) decreases (e.g., is less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., pancreatic acinar cells) increases (e.g., is more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two detections, the subject undergoes treatment, and thus the detection results indicate the treatment effect.

[0533] In one embodiment, a method is also provided for determining the cell type of disease cells (e.g., cancer cells), the primary origin of the disease (e.g., cancer cells), or the signal or source of the disease (e.g., cancer cells). In some embodiments, the primary origin of the cancer cells is unknown. In some embodiments, the methods described above include detecting the methylation status of one or more DNA fragments of the cancer cells, and the methylation status can then be used to determine that the cells are pancreatic acinar cells, as described above.

[0534] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus cell type detection can help associate the genetic variations with cancer. In some embodiments, the genetic variations include mutations. In some embodiments, the genetic variations include deletions or insertions. In some embodiments, the genetic variations constitute microsatellite instability. In some embodiments, the genetic variations constitute loss of heterozygosity. In some embodiments, the genetic variations disrupt or alter gene splicing. In some embodiments, the genetic variations cause frameshifts or generate premature stop codons. Once the primary origin of the cancer is identified, the subject can be treated with an appropriate regimen for that cancer type.

[0535] A9. Pancreatic Alpha Cells

[0536] Also as provided in Table A, compared to all other cell types in humans, some genomic locations in pancreatic alpha cells are consistently undermethylated or overmethylated.

[0537] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from pancreatic alpha cells is provided. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more) CpG sites in a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten, or all) of the CpG sites is located within a human genomic sequence selected from SEQ ID NO: 1181-1198, 1199-1282, 1283-1284, 1285-1287, 1288-1292, or 1293-1306, or within a human genomic sequence selected from SEQ ID NO: 1181-1198 or 1199-1282, or is less than 100 bp, 200 bp, 500 bp, or 1 kb away from the foregoing human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from pancreatic alpha cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from pancreatic alpha cells. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from pancreatic alpha cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from pancreatic alpha cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from pancreatic alpha cells.

[0538] In some embodiments, the method requires detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites in a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites is located within a human genomic sequence selected from SEQ ID NOs: 1307-1315, 1316-1316 or 1317-1331 or within a human genomic sequence selected from SEQ ID NOs: 1307-1315, or is less than 100 bp, 200 bp, 500 bp or 1 kb away from the foregoing human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from pancreatic alpha cells. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from pancreatic alpha cells. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from pancreatic alpha cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from pancreatic alpha cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from pancreatic alpha cells.

[0539] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by genomic sequences SEQ ID NOs: 1181-1198, 1199-1282, 1283-1284, 1285-1287, 1288-1292, 1293-1306, 1307-1315, 1316-1316 or 1317-1331.

[0540] The cell type identification method can be used to detect diseases or conditions associated with cell type. In one embodiment, when cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva or cerebrospinal fluid) is identified as being from pancreatic alpha cells of a subject, the method indicates that the subject has abnormal cell death and / or has a disease associated with such cells. In some embodiments, the disease or condition is damage, inflammation or cancer of pancreatic alpha cells. In some embodiments, the disease is diabetes.

[0541] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., pancreatic alpha cells) decreases (e.g., is less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., pancreatic alpha cells) increases (e.g., is more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two tests, the subject undergoes treatment, so the test results indicate the treatment effect.

[0542] In one embodiment, a method is also provided for determining the cell type of disease cells (e.g., cancer cells), the primary origin of the disease (e.g., cancer cells), or the signal or origin of the disease (e.g., cancer cells). In some embodiments, the primary origin of the cancer cells is unknown. In some embodiments, the methods described above include detecting the methylation status of one or more DNA fragments of the cancer cells, and the methylation status can then be used to determine that the cells are pancreatic alpha cells, as described above.

[0543] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, so cell type detection can help associate the genetic variations with cancer. In some embodiments, the genetic variations include mutations. In some embodiments, the genetic variations include deletions or insertions. In some embodiments, the genetic variations constitute microsatellite instability. In some embodiments, the genetic variations constitute loss of heterozygosity. In some embodiments, the genetic variations disrupt or alter gene splicing. In some embodiments, the genetic variations cause a frameshift or generate a premature stop codon. Once the primary origin of the cancer is identified, the subject can be treated with an appropriate regimen for that cancer type.

[0544] A10. Pancreatic Beta Fine Cells

[0545] Also as provided in Table A, compared to all other cell types in humans, some genomic locations in pancreatic beta cells are consistently undermethylated or overmethylated.

[0546] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from pancreatic beta cells is provided. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites is located within a human genomic sequence selected from SEQ ID NO: 1332 - 1351, 1352 - 1440, 1441 - 1445 or 1446 - 1460 or selected from SEQ ID NO: 1332 - 1351 or 1352 - 1440, or is less than 100 bp, 200 bp, 500 bp or 1 kb away from the aforementioned human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from pancreatic beta cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from pancreatic beta cells. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from pancreatic beta cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from pancreatic beta cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from pancreatic beta cells.

[0547] In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites in a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites are within a human genomic sequence selected from SEQ ID NOs: 1461 - 1471 or 1472 - 1485 or within a human genomic sequence selected from SEQ ID NOs: 1461 - 1471, or are less than 100 bp, 200 bp, 500 bp or 1 kb away from the aforementioned human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from pancreatic beta cells. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from pancreatic beta cells. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from pancreatic beta cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from pancreatic beta cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from pancreatic beta cells.

[0548] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by the genomic sequences SEQ ID NOs: 1332 - 1351, 1352 - 1440, 1441 - 1445, 1446 - 1460, 1461 - 1471 or 1472 - 1485.

[0549] The cell type identification method can be used to detect diseases or conditions associated with cell type. In one embodiment, when cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva or cerebrospinal fluid) is identified as being from the pancreatic beta cells of a subject, the method indicates that the subject has abnormal cell death and / or has a disease associated with that cell. In some embodiments, the disease or condition is damage, inflammation or cancer of pancreatic beta cells. In some embodiments, the disease is diabetes.

[0550] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., pancreatic beta cells) decreases (e.g., is less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., pancreatic beta cells) increases (e.g., is more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two detections, the subject undergoes treatment, so the detection results indicate the treatment effect.

[0551] In one embodiment, a method is also provided for determining the cell type of a disease cell (e.g., a cancer cell), the primary origin of a disease (e.g., a cancer cell), or the signal or source of a disease (e.g., a cancer cell). In some embodiments, the primary origin of the cancer cell is unknown. In some embodiments, the methods described above include detecting the methylation status of one or more DNA fragments of the cancer cell, and the methylation status can then be used to determine that the cell is a pancreatic beta cell, as described above.

[0552] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of the cancer cells. In some embodiments, genetic variations can also be present in the DNA fragments, so cell type detection can help to associate the genetic variations with cancer. In some embodiments, the genetic variations include mutations. In some embodiments, the genetic variations include deletions or insertions. In some embodiments, the genetic variations constitute microsatellite instability. In some embodiments, the genetic variations constitute loss of heterozygosity. In some embodiments, the genetic variations disrupt or alter gene splicing. In some embodiments, the genetic variations cause a frameshift or generate a premature stop codon. Once the primary origin of the cancer is identified, the subject can be treated with an appropriate regimen for that cancer type.

[0553] A11. Pancreatic Delta Cells

[0554] Also as provided in Table A, compared to all other cell types in humans, some genomic locations in pancreatic delta cells are consistently undermethylated or overmethylated.

[0555] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from pancreatic delta cells is provided. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more) CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten, or all) of the CpG sites is located within a human genomic sequence selected from SEQ ID NO: 1486 - 1508, 1509 - 1594, 1595 - 1596, 1597 - 1598, or 1599 - 1613, or within a human genomic sequence selected from SEQ ID NO: 1486 - 1508 or 1509 - 1594, or is less than 100 bp, 200 bp, 500 bp, or 1 kb away from the aforementioned human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from pancreatic delta cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from pancreatic delta cells. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from pancreatic delta cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from pancreatic delta cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from pancreatic delta cells.

[0556] In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites in a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites are located within a human genomic sequence selected from SEQ ID NOs: 1614-1624, 1625-1625 or 1626-1638 or within a human genomic sequence selected from SEQ ID NOs: 1614-1624, or are within less than 100 bp, 200 bp, 500 bp or 1 kb of the foregoing human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from pancreatic delta cells. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from pancreatic delta cells. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from pancreatic delta cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from pancreatic delta cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from pancreatic delta cells.

[0557] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by the genomic sequences SEQ ID NOs: 1486-1508, 1509-1594, 1595-1596, 1597-1598, 1599-1613, 1614-1624, 1625-1625 or 1626-1638.

[0558] The cell type identification method can be used to detect diseases or conditions associated with a cell type. In one embodiment, when cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva or cerebrospinal fluid) is identified as being from pancreatic delta cells of a subject, the method indicates that the subject has abnormal cell death and / or has a disease associated with that cell. In some embodiments, the disease or condition is injury, inflammation or cancer of pancreatic delta cells. In some embodiments, the disease is diabetes.

[0559] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., pancreatic delta cells) is decreased (e.g., less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., pancreatic delta cells) is increased (e.g., more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two tests, the subject undergoes treatment, and thus the test results indicate the treatment effect.

[0560] In one embodiment, a method is also provided for determining the cell type of disease cells (e.g., cancer cells), the primary origin of the disease (e.g., cancer cells), or the signal or source of the disease (e.g., cancer cells). In some embodiments, the primary origin of the cancer cells is unknown. In some embodiments, the methods described above include detecting the methylation status of one or more DNA fragments of the cancer cells, and the methylation status can then be used to determine that the cells are pancreatic delta cells, as described above.

[0561] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of the cancer cells. In some embodiments, genetic variations can also be present in the DNA fragments, and thus cell type detection can help to associate the genetic variations with cancer. In some embodiments, the genetic variations include mutations. In some embodiments, the genetic variations include deletions or insertions. In some embodiments, the genetic variations constitute microsatellite instability. In some embodiments, the genetic variations constitute loss of heterozygosity. In some embodiments, the genetic variations disrupt or alter gene splicing. In some embodiments, the genetic variations cause a frameshift or generate a premature stop codon. Once the primary origin of the cancer is identified, the subject can be treated with an appropriate regimen for that cancer type.

[0562] A12. Pancreatic Duct Cells

[0563] Also as provided in Table A, some genomic locations in pancreatic duct cells are consistently hypomethylated or hypermethylated compared to all other cell types in humans.

[0564] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from pancreatic duct cells is provided. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites is located within a human genomic sequence selected from SEQ ID NO: 1639 - 1658, 1659 - 1742, 1743 - 1743, 1744 - 1747, 1748 - 1751 or 1752 - 1767 or selected from SEQ ID NO: 1639 - 1658 or 1659 - 1742, or is within less than 100 bp, 200 bp, 500 bp or 1 kb of the foregoing human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from pancreatic duct cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from pancreatic duct cells. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from pancreatic duct cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from pancreatic duct cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from pancreatic duct cells.

[0565] In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more) CpG sites in a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten, or all) of the CpG sites are located within a human genomic sequence selected from SEQ ID NO: 1768 - 1779 or 1780 - 1792 or within a human genomic sequence selected from SEQ ID NO: 1768 - 1779, or within 100 bp, 200 bp, 500 bp, or 1 kb of the foregoing human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method subsequently identifies that the target DNA fragment is from pancreatic duct cells. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from pancreatic duct cells. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from pancreatic duct cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from pancreatic duct cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from pancreatic duct cells.

[0566] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by the genomic sequences SEQ ID NO: 1639 - 1658, 1659 - 1742, 1743 - 1743, 1744 - 1747, 1748 - 1751, 1752 - 1767, 1768 - 1779, or 1780 - 1792.

[0567] The cell type identification method can be used to detect diseases or conditions associated with cell type. In one embodiment, when cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva, or cerebrospinal fluid) is identified as being from the pancreatic duct cells of a subject, the method indicates that the subject has abnormal cell death and / or has a disease associated with that cell. In some embodiments, the disease or condition is injury, inflammation, or cancer of pancreatic duct cells. In some embodiments, the disease is diabetes.

[0568] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., pancreatic duct cells) decreases (e.g., is less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., pancreatic duct cells) increases (e.g., is more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two detections, the subject undergoes treatment, so the detection results indicate the treatment effect.

[0569] In one embodiment, a method for determining the cell type of disease cells (e.g., cancer cells), the primary origin of the disease (e.g., cancer cells), or the signal or source of the disease (e.g., cancer cells) is also provided. In some embodiments, the primary origin of the cancer cells is unknown. In some embodiments, the methods described above include detecting the methylation status of one or more DNA fragments of the cancer cells, and the methylation status can then be used to determine that the cells are pancreatic duct cells, as described above.

[0570] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of the cancer cells. In some embodiments, genetic variations can also be present in the DNA fragments, so cell type detection can help associate the genetic variations with cancer. In some embodiments, the genetic variations include mutations. In some embodiments, the genetic variations include deletions or insertions. In some embodiments, the genetic variations constitute microsatellite instability. In some embodiments, the genetic variations constitute loss of heterozygosity. In some embodiments, the genetic variations disrupt or alter gene splicing. In some embodiments, the genetic variations cause a frameshift or generate a premature stop codon. Once the primary origin of the cancer is identified, the subject can be treated with an appropriate regimen for that cancer type.

[0571] Group 1 - GI Epithelium (Colonic Epithelium, Gastric Epithelium, and Small Intestinal Epithelium)

[0572] Also as provided in Table A, compared to all other cell types in humans, a group of cells, namely the colon epithelium, the gastric epithelium, and some genomic locations in the small intestine epithelium, are hypomethylated or hypermethylated consistently.

[0573] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from cells selected from colonic epithelium, gastric epithelium, and small intestinal epithelium is provided. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more) CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten, or all) of the CpG sites is located within a human genomic sequence selected from SEQ ID NO: 6541 - 6556, 6557 - 6557, or 6558 - 6565, or from SEQ ID NO: 6541 - 6556, or is within less than 100 bp, 200 bp, 500 bp, or 1 kb of the foregoing human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from cells selected from colonic epithelium, gastric epithelium, and small intestinal epithelium. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from cells selected from colonic epithelium, gastric epithelium, and small intestinal epithelium. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from cells selected from colonic epithelium, gastric epithelium, and small intestinal epithelium. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from cells selected from colonic epithelium, gastric epithelium, and small intestinal epithelium. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from cells selected from colonic epithelium, gastric epithelium, and small intestinal epithelium.

[0574] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine the cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by the genomic sequences SEQ ID NO: 6541 - 6556, 6557 - 6557, or 6558 - 6565.

[0575] Cell type identification methods can be used to detect diseases or conditions associated with cell types. In one embodiment, when cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva, or cerebrospinal fluid) is identified as being from cells selected from the colonic epithelium, gastric epithelium, and small intestinal epithelium of a subject, the method indicates that the subject has abnormal cell death and / or has a disease associated with that cell. In some embodiments, the disease or condition is damage, inflammation, or cancer of cells selected from the colonic epithelium, gastric epithelium, and small intestinal epithelium.

[0576] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., cells selected from the colonic epithelium, gastric epithelium, and small intestinal epithelium) is decreased (e.g., less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., cells selected from the colonic epithelium, gastric epithelium, and small intestinal epithelium) is increased (e.g., more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two detections, the subject undergoes treatment, such that the detection results indicate the treatment effect.

[0577] In one embodiment, there is also provided a method for determining the cell type of disease cells (e.g., cancer cells), the primary origin of a disease (e.g., cancer cells), or the signal or source of a disease (e.g., cancer cells). In some embodiments, the primary origin of the cancer cells is unknown. In some embodiments, the methods described above include detecting the methylation status of one or more DNA fragments of the cancer cells, and then the methylation status can be used to determine that the cells are cells selected from the colonic epithelium, gastric epithelium, and small intestinal epithelium, as described above.

[0578] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus cell type detection can help correlate the genetic variations with cancer. In some embodiments, the genetic variations include mutations. In some embodiments, the genetic variations include deletions or insertions. In some embodiments, the genetic variations constitute microsatellite instability. In some embodiments, the genetic variations constitute loss of heterozygosity. In some embodiments, the genetic variations disrupt or alter gene splicing. In some embodiments, the genetic variations cause a frameshift or generate a premature stop codon. Once the primary source of the cancer has been identified, the subject can be treated with an appropriate regimen for that cancer type.

[0579] Group 2 - Small Intestinal Epithelium and Colonic Epithelium

[0580] Also as provided in Table A, compared to all other cell types in humans, a group of cells, namely the small intestinal epithelium and some genomic locations in the colonic epithelium, are consistently hypomethylated or hypermethylated.

[0581] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from cells selected from small intestinal epithelium and colonic epithelium is provided. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more) CpG sites in a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten, or all) of the CpG sites is located within a human genomic sequence selected from SEQ ID NO: 6695 - 6702, 6703 - 6760, 6761 - 6777, or 6778 - 6820, or within a human genomic sequence selected from SEQ ID NO: 6695 - 6702 or 6703 - 6760, or within 100 bp, 200 bp, 500 bp, or 1 kb of the foregoing human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from cells selected from small intestinal epithelium and colonic epithelium. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from cells selected from small intestinal epithelium and colonic epithelium. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from cells selected from small intestinal epithelium and colonic epithelium. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from cells selected from small intestinal epithelium and colonic epithelium. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from cells selected from small intestinal epithelium and colonic epithelium.

[0582] In some embodiments, the method requires detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites in a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) CpG sites are located within a human genomic sequence selected from SEQ ID NO: 6821 - 6825 or 6826 - 6845 or within a human genomic sequence selected from SEQ ID NO: 6821 - 6825, or within 100 bp, 200 bp, 500 bp or 1 kb of the foregoing human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from a cell selected from small intestinal epithelium and colonic epithelium. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from a cell selected from small intestinal epithelium and colonic epithelium. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from a cell selected from small intestinal epithelium and colonic epithelium. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from a cell selected from small intestinal epithelium and colonic epithelium. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from a cell selected from small intestinal epithelium and colonic epithelium.

[0583] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by genomic sequences SEQ ID NO: 6695 - 6702, 6703 - 6760, 6761 - 6777, 6778 - 6820, 6821 - 6825 or 6826 - 6845.

[0584] The cell type identification method can be used to detect diseases or conditions associated with cell type. In one embodiment, when cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva or cerebrospinal fluid) is identified as being from a cell selected from small intestinal epithelium and colonic epithelium of a subject, the method indicates that the subject has abnormal cell death and / or suffers from a disease associated with that cell. In some embodiments, the disease or condition is injury, inflammation or cancer of a cell selected from small intestinal epithelium and colonic epithelium.

[0585] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., cells selected from small intestinal epithelium and colonic epithelium) decreases (e.g., is less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., cells selected from small intestinal epithelium and colonic epithelium) increases (e.g., is more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two tests, the subject undergoes treatment, and thus the test results indicate the treatment effect.

[0586] In one embodiment, a method for determining the cell type of disease cells (e.g., cancer cells), the primary source of the disease (e.g., cancer cells), or the signal or origin of the disease (e.g., cancer cells) is also provided. In some embodiments, the primary source of the cancer cells is unknown. In some embodiments, the methods described above include detecting the methylation status of one or more DNA fragments of the cancer cells, and the methylation status can then be used to determine that the cells are cells selected from small intestinal epithelium and colonic epithelium, as described above.

[0587] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of the cancer cells. In some embodiments, genetic variations can also be present in the DNA fragments, and thus cell type detection can help to associate the genetic variations with cancer. In some embodiments, the genetic variations include mutations. In some embodiments, the genetic variations include deletions or insertions. In some embodiments, the genetic variations constitute microsatellite instability. In some embodiments, the genetic variations constitute loss of heterozygosity. In some embodiments, the genetic variations disrupt or alter gene splicing. In some embodiments, the genetic variations cause a frameshift or generate a premature stop codon. Once the primary source of the cancer is identified, the subject can be treated with an appropriate regimen for that cancer type.

[0588] Group 3 - Gastric Epithelium and Small Intestinal Epithelium

[0589] Also as provided in Table A, compared to all other cell types in humans, some genomic positions in a group of cells, namely gastric epithelium and small intestinal epithelium, are consistently hypomethylated or hypermethylated.

[0590] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from cells selected from gastric epithelium and small intestinal epithelium is provided. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites are located within a human genomic sequence selected from SEQ ID NO: 6566 - 6589, 6590 - 6672, 6673 - 6673, 6674 - 6674 or 6675 - 6690 or selected from SEQ ID NO: 6566 - 6589 or 6590 - 6672, or are less than 100 bp, 200 bp, 500 bp or 1 kb away from the foregoing human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from cells selected from gastric epithelium and small intestinal epithelium. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from cells selected from gastric epithelium and small intestinal epithelium. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from cells selected from gastric epithelium and small intestinal epithelium. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from cells selected from gastric epithelium and small intestinal epithelium. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from cells selected from gastric epithelium and small intestinal epithelium.

[0591] In some embodiments, the method requires detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites are within the human genomic sequence selected from SEQ ID NO: 6691 or 6692 - 6694 or are within 100 bp, 200 bp, 500 bp or 1 kb of the foregoing human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from cells selected from gastric epithelium and small intestinal epithelium. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from cells selected from gastric epithelium and small intestinal epithelium. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from cells selected from gastric epithelium and small intestinal epithelium. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from cells selected from gastric epithelium and small intestinal epithelium. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from cells selected from gastric epithelium and small intestinal epithelium.

[0592] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by the genomic sequences SEQ ID NO: 6566 - 6589, 6590 - 6672, 6673 - 6673, 6674 - 6674, 6675 - 6690, 6691 or 6692 - 6694.

[0593] The cell type identification method can be used to detect diseases or conditions associated with cell type. In one embodiment, when cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva or cerebrospinal fluid) is identified as being from cells selected from the gastric epithelium and small intestinal epithelium of a subject, the method indicates that the subject has abnormal cell death and / or suffers from a disease associated with such cells. In some embodiments, the disease or condition is injury, inflammation or cancer of cells selected from gastric epithelium and small intestinal epithelium.

[0594] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., cells selected from gastric epithelium and small intestinal epithelium) decreases (e.g., is less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., cells selected from gastric epithelium and small intestinal epithelium) increases (e.g., is more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two tests, the subject undergoes treatment, and thus the test results indicate the treatment effect.

[0595] In one embodiment, a method is also provided for determining the cell type of disease cells (e.g., cancer cells), the primary source of the disease (e.g., cancer cells), or the signal or origin of the disease (e.g., cancer cells). In some embodiments, the primary source of the cancer cells is unknown. In some embodiments, the methods described above include detecting the methylation status of one or more DNA fragments of the cancer cells, and the methylation status can then be used to determine that the cells are cells selected from gastric epithelium and small intestinal epithelium, as described above.

[0596] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus cell type detection can help to associate the genetic variations with cancer. In some embodiments, the genetic variations include mutations. In some embodiments, the genetic variations include deletions or insertions. In some embodiments, the genetic variations constitute microsatellite instability. In some embodiments, the genetic variations constitute loss of heterozygosity. In some embodiments, the genetic variations disrupt or alter gene splicing. In some embodiments, the genetic variations cause a frameshift or generate a premature stop codon. Once the primary source of the cancer is identified, the subject can be treated with an appropriate regimen for that cancer type.

[0597] Group 4 - Colonic Fibroblasts and Cardiac Fibroblasts

[0598] Also as provided in Table A, compared to all other cell types in humans, some genomic positions in a group of cells, namely colon fibroblasts and heart fibroblasts, are consistently hypomethylated or hypermethylated.

[0599] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from a cell selected from colon fibroblasts and cardiac fibroblasts is provided. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites in a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites is located within a human genomic sequence selected from SEQ ID NO: 6846-6863, 6864-6869, 6870-6872, 6873-6876 or 6877-6878 or selected from SEQ ID NO: 6846-6863 or 6864-6869, or is less than 100 bp, 200 bp, 500 bp or 1 kb away from the aforementioned human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from a cell selected from colon fibroblasts and cardiac fibroblasts. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from a cell selected from colon fibroblasts and cardiac fibroblasts. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from a cell selected from colon fibroblasts and cardiac fibroblasts. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from a cell selected from colon fibroblasts and cardiac fibroblasts. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from a cell selected from colon fibroblasts and cardiac fibroblasts.

[0600] In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites in a target DNA fragment in a biological sample, where at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites are located within a human genomic sequence selected from SEQ ID NO: 6879 - 6890 or 6891 - 6898 or within a human genomic sequence selected from SEQ ID NO: 6879 - 6890, or are less than 100 bp, 200 bp, 500 bp or 1 kb away from the aforementioned human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from a cell selected from colon fibroblasts and heart fibroblasts. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from a cell selected from colon fibroblasts and heart fibroblasts. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from a cell selected from colon fibroblasts and heart fibroblasts. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from a cell selected from colon fibroblasts and heart fibroblasts. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from a cell selected from colon fibroblasts and heart fibroblasts.

[0601] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by genomic sequences SEQ ID NO: 6846 - 6863, 6864 - 6869, 6870 - 6872, 6873 - 6876, 6877 - 6878, 6879 - 6890 or 6891 - 6898.

[0602] A method for cell type identification can be used to detect diseases or conditions associated with a cell type. In one embodiment, when cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva, or cerebrospinal fluid) is identified as being from a cell selected from a subject's colonic fibroblasts and cardiac fibroblasts, the method indicates that the subject has abnormal cell death and / or is suffering from a disease associated with that cell. In some embodiments, the disease or condition is damage, inflammation, or cancer of a cell selected from colonic fibroblasts and cardiac fibroblasts.

[0603] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., a cell selected from colonic fibroblasts and cardiac fibroblasts) decreases (e.g., is less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the above method includes making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., a cell selected from colonic fibroblasts and cardiac fibroblasts) increases (e.g., is more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the above method includes making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two detections, the subject undergoes treatment, so the detection results indicate the treatment effect.

[0604] In one embodiment, a method is also provided for determining the cell type of disease cells (e.g., cancer cells), the primary source of a disease (e.g., cancer cells), or the signal or origin of a disease (e.g., cancer cells). In some embodiments, the primary source of the cancer cells is unknown. In some embodiments, the above method includes detecting the methylation status of one or more DNA fragments of the cancer cells, and then the methylation status can be used to determine that the cell is a cell selected from colonic fibroblasts and cardiac fibroblasts, as described above.

[0605] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of the cancer cells. In some embodiments, genetic variations can also be present in the DNA fragments, and thus cell type detection can help to correlate the genetic variations with cancer. In some embodiments, the genetic variations include mutations. In some embodiments, the genetic variations include deletions or insertions. In some embodiments, the genetic variations constitute microsatellite instability. In some embodiments, the genetic variations constitute loss of heterozygosity. In some embodiments, the genetic variations disrupt or alter gene splicing. In some embodiments, the genetic variations cause frameshifts or generate premature stop codons. Once the primary source of the cancer has been identified, the subject can be treated with an appropriate regimen for that cancer type.

[0606] Group 5 - Pancreatic Alpha, Beta, and Delta Cells

[0607] Also as provided in Table A, some genomic locations are consistently hypomethylated or hypermethylated in a group of cells, namely pancreatic alpha, beta, and delta cells, compared to all other cell types in humans.

[0608] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from cells selected from pancreatic alpha, beta, and delta cells is provided. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more) CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten, or all) of the CpG sites is located within a human genomic sequence selected from SEQ ID NO: 5924 - 5935, 5936 - 6011, 6012 - 6012, 6013 - 6014, 6015 - 6026, or 6027 - 6050, or from SEQ ID NO: 5924 - 5935 or 5936 - 6011, or is within less than 100 bp, 200 bp, 500 bp, or 1 kb of the foregoing human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from cells selected from pancreatic alpha, beta, and delta cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from cells selected from pancreatic alpha, beta, and delta cells. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from cells selected from pancreatic alpha, beta, and delta cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from cells selected from pancreatic alpha, beta, and delta cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from cells selected from pancreatic alpha, beta, and delta cells.

[0609] In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites in a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites are within a human genomic sequence selected from SEQ ID NO: 6051 - 6057 or 6058 - 6075 or within a human genomic sequence selected from SEQ ID NO: 6051 - 6057, or are within 100 bp, 200 bp, 500 bp or 1 kb of the foregoing human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from a cell selected from pancreatic alpha, beta, and delta cells. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from a cell selected from pancreatic alpha, beta, and delta cells. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from a cell selected from pancreatic alpha, beta, and delta cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from a cell selected from pancreatic alpha, beta, and delta cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from a cell selected from pancreatic alpha, beta, and delta cells.

[0610] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by the genomic sequences SEQ ID NO: 5924 - 5935, 5936 - 6011, 6012 - 6012, 6013 - 6014, 6015 - 6026, 6027 - 6050, 6051 - 6057 or 6058 - 6075.

[0611] Cell type identification methods can be used to detect diseases or conditions associated with cell types. In one embodiment, when cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva, or cerebrospinal fluid) is identified as being from cells selected from a subject's pancreatic alpha, beta, and delta cells, the method indicates that the subject has abnormal cell death and / or has a disease associated with that cell. In some embodiments, the disease or condition is damage, inflammation, or cancer of a cell selected from pancreatic alpha, beta, and delta cells.

[0612] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., cells selected from pancreatic alpha, beta, and delta cells) is decreased (e.g., less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., cells selected from pancreatic alpha, beta, and delta cells) is increased (e.g., more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two detections, the subject undergoes treatment, and thus the detection results indicate the treatment effect.

[0613] In one embodiment, a method is also provided for determining the cell type of disease cells (e.g., cancer cells), the primary source of the disease (e.g., cancer cells), or the signal or origin of the disease (e.g., cancer cells). In some embodiments, the primary source of the cancer cells is unknown. In some embodiments, the methods described above include detecting the methylation status of one or more DNA fragments of the cancer cells, and the methylation status can then be used to determine that the cells are cells selected from pancreatic alpha, beta, and delta cells, as described above.

[0614] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus cell type detection can help to associate the genetic variations with cancer. In some embodiments, the genetic variations include mutations. In some embodiments, the genetic variations include deletions or insertions. In some embodiments, the genetic variations constitute microsatellite instability. In some embodiments, the genetic variations constitute loss of heterozygosity. In some embodiments, the genetic variations disrupt or alter gene splicing. In some embodiments, the genetic variations cause a frameshift or generate a premature stop codon. Once the primary source of the cancer has been identified, the subject can be treated with an appropriate regimen for that cancer type.

[0615] B. Reproductive and Urinary Tract Cells

[0616] B1. Endometrial Epithelium

[0617] Also as provided in Table A, some genomic locations in endometrial epithelial cells are consistently hypomethylated or hypermethylated compared to all other cell types in humans.

[0618] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from endometrial epithelial cells is provided. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites is located within a human genomic sequence selected from SEQ ID NO: 1793 - 1864, 1865 - 1872 or 1873 - 1892 or within a human genomic sequence selected from SEQ ID NO: 1793 - 1864, or is less than 100 bp, 200 bp, 500 bp or 1 kb away from the aforementioned human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from endometrial epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from endometrial epithelial cells. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from endometrial epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from endometrial epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from endometrial epithelial cells.

[0619] In some embodiments, the method requires detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) CpG sites are located within a human genomic sequence selected from SEQ ID NO: 1893 - 1905 or 1906 - 1917 or within a human genomic sequence selected from SEQ ID NO: 1893 - 1905, or are less than 100 bp, 200 bp, 500 bp or 1 kb away from the foregoing human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from endometrial epithelial cells. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from endometrial epithelial cells. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from endometrial epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from endometrial epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from endometrial epithelial cells.

[0620] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by the genomic sequences SEQ ID NO: 1793 - 1864, 1865 - 1872, 1873 - 1892, 1893 - 1905 or 1906 - 1917.

[0621] The cell type identification method can be used to detect diseases or conditions associated with cell type. In one embodiment, when cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva or cerebrospinal fluid) is identified as being from the endometrial epithelial cells of a subject, the method indicates that the subject has abnormal cell death and / or has a disease associated with that cell. In some embodiments, the disease or condition is damage, inflammation or cancer of the endometrial epithelium.

[0622] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., endometrial epithelial cells) decreases (e.g., is less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., endometrial epithelial cells) increases (e.g., is more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two tests, the subject undergoes treatment, and thus the test results indicate the treatment effect.

[0623] In one embodiment, a method is also provided for determining the cell type of disease cells (e.g., cancer cells), the primary origin of the disease (e.g., cancer cells), or the signal or source of the disease (e.g., cancer cells). In some embodiments, the primary origin of the cancer cells is unknown. In some embodiments, the methods described above include detecting the methylation status of one or more DNA fragments of the cancer cells, and the methylation status can then be used to determine that the cells are endometrial epithelial cells, as described above.

[0624] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of the cancer cells. In some embodiments, genetic variations can also be present in the DNA fragments, and thus cell type detection can help associate the genetic variations with cancer. In some embodiments, the genetic variations include mutations. In some embodiments, the genetic variations include deletions or insertions. In some embodiments, the genetic variations constitute microsatellite instability. In some embodiments, the genetic variations constitute loss of heterozygosity. In some embodiments, the genetic variations disrupt or alter gene splicing. In some embodiments, the genetic variations cause a frameshift or generate a premature stop codon. Once the primary origin of the cancer is identified, the subject can be treated with an appropriate regimen for that cancer type.

[0625] B2. Oviductal Epithelium

[0626] Also as provided in Table A, some genomic locations in fallopian tube epithelial cells are consistently undermethylated or overmethylated compared to all other cell types in humans.

[0627] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from fallopian tube epithelial cells is provided. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites is located within a human genomic sequence selected from SEQ ID NO: 1918-1937, 1938-2022, 2023-2024, 2025-2029 or 2030-2042 or selected from SEQ ID NO: 1918-1937 or 1938-2022, or is less than 100 bp, 200 bp, 500 bp or 1 kb away from the aforementioned human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from fallopian tube epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from fallopian tube epithelial cells. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from fallopian tube epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from fallopian tube epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from fallopian tube epithelial cells.

[0628] In some embodiments, the method requires detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites in a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) CpG sites are located within a human genomic sequence selected from SEQ ID NO: 2043 - 2061 or 2062 - 2067 or within a human genomic sequence selected from SEQ ID NO: 2043 - 2061, or within less than 100 bp, 200 bp, 500 bp or 1 kb from the foregoing human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from fallopian tube epithelial cells. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from fallopian tube epithelial cells. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from fallopian tube epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from fallopian tube epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from fallopian tube epithelial cells.

[0629] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by genomic sequences SEQ ID NO: 1918 - 1937, 1938 - 2022, 2023 - 2024, 2025 - 2029, 2030 - 2042, 2043 - 2061 or 2062 - 2067.

[0630] The cell type identification method can be used to detect diseases or conditions associated with a cell type. In one embodiment, when cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva or cerebrospinal fluid) is identified as being from the fallopian tube epithelial cells of a subject, the method indicates that the subject has abnormal cell death and / or suffers from a disease associated with that cell. In some embodiments, the disease or condition is injury, inflammation or cancer of the fallopian tube epithelium.

[0631] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., fallopian tube epithelial cells) decreases (e.g., is less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., fallopian tube epithelial cells) increases (e.g., is more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two tests, the subject undergoes treatment, so the test results indicate the treatment effect.

[0632] In one embodiment, a method is also provided for determining the cell type of disease cells (e.g., cancer cells), the primary origin of the disease (e.g., cancer cells), or the signal or source of the disease (e.g., cancer cells). In some embodiments, the primary origin of the cancer cells is unknown. In some embodiments, the methods described above include detecting the methylation status of one or more DNA fragments of the cancer cells, and the methylation status can then be used to determine that the cells are fallopian tube epithelial cells, as described above.

[0633] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, so cell type detection can help associate the genetic variations with cancer. In some embodiments, the genetic variations include mutations. In some embodiments, the genetic variations include deletions or insertions. In some embodiments, the genetic variations constitute microsatellite instability. In some embodiments, the genetic variations constitute loss of heterozygosity. In some embodiments, the genetic variations disrupt or alter gene splicing. In some embodiments, the genetic variations cause a frameshift or generate a premature stop codon. Once the primary origin of the cancer is identified, the subject can be treated with an appropriate regimen for that cancer type.

[0634] B3. Renal Epithelium

[0635] Also as provided in Table A, some genomic locations in renal epithelial cells are consistently undermethylated or overmethylated compared to all other cell types in humans.

[0636] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from renal epithelial cells is provided. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites is located within a human genomic sequence selected from SEQ ID NO: 2068 - 2080, 2081 - 2141, 2142 - 2144, 2145 - 2156 or 2157 - 2194, or is less than 100 bp, 200 bp, 500 bp or 1 kb away from the aforementioned human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from renal epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from renal epithelial cells. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from renal epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from renal epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from renal epithelial cells.

[0637] In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites in a target DNA fragment in a biological sample, where at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites are located within a human genomic sequence selected from SEQ ID NO: 2195 - 2209 or 2210 - 2219 or within a human genomic sequence selected from SEQ ID NO: 2195 - 2209, or within 100 bp, 200 bp, 500 bp or 1 kb of the foregoing human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from renal epithelial cells. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from renal epithelial cells. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from renal epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from renal epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from renal epithelial cells.

[0638] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by genomic sequences SEQ ID NO: 2068 - 2080, 2081 - 2141, 2142 - 2144, 2145 - 2156, 2157 - 2194, 2195 - 2209 or 2210 - 2219.

[0639] The cell type identification method can be used to detect diseases or conditions associated with a cell type. In one embodiment, when cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva or cerebrospinal fluid) is identified as being from renal epithelial cells of a subject, the method indicates that the subject has abnormal cell death and / or has a disease associated with that cell. In some embodiments, the disease or condition is damage, inflammation or cancer of the renal epithelium.

[0640] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., renal epithelial cells) decreases (e.g., is less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the method described above includes making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., renal epithelial cells) increases (e.g., is more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the method described above includes making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two detections, the subject undergoes treatment, so the detection results indicate the treatment effect.

[0641] In one embodiment, a method for determining the cell type of disease cells (e.g., cancer cells), the primary origin of the disease (e.g., cancer cells), or the signal or source of the disease (e.g., cancer cells) is also provided. In some embodiments, the primary origin of the cancer cells is unknown. In some embodiments, the method described above includes detecting the methylation status of one or more DNA fragments of the cancer cells, and then the methylation status can be used to determine that the cells are renal epithelial cells, as described above.

[0642] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of cancer cells. In some embodiments, genetic variations can also be present in the DNA fragments, so cell type detection can help associate the genetic variations with cancer. In some embodiments, the genetic variations include mutations. In some embodiments, the genetic variations include deletions or insertions. In some embodiments, the genetic variations constitute microsatellite instability. In some embodiments, the genetic variations constitute loss of heterozygosity. In some embodiments, the genetic variations disrupt or alter gene splicing. In some embodiments, the genetic variations cause frameshifts or generate premature stop codons. Once the primary origin of the cancer is identified, the subject can be treated with an appropriate regimen for that cancer type.

[0643] B4. Bladder Epithelium

[0644] Also as provided in Table A, some genomic locations in bladder epithelial cells are consistently undermethylated or overmethylated compared to all other cell types in humans.

[0645] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from bladder epithelial cells is provided. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites is located within a human genomic sequence selected from SEQ ID NO: 2220-2233, 2234-2298, 2299-2299, 2300-2303, 2304-2313 or 2314-2345 or selected from SEQ ID NO: 2220-2233 or 2234-2298, or is less than 100 bp, 200 bp, 500 bp or 1 kb away from the aforementioned human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from bladder epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from bladder epithelial cells. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from bladder epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from bladder epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from bladder epithelial cells.

[0646] In some embodiments, the method requires detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) CpG sites are located within a human genomic sequence selected from SEQ ID NO: 2346-2350, 2351-2351 or 2352-2370 or selected from SEQ ID NO: 2346-2350, or are less than 100 bp, 200 bp, 500 bp or 1 kb away from the aforementioned human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from bladder epithelial cells. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from bladder epithelial cells. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from bladder epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from bladder epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from bladder epithelial cells.

[0647] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine the cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by the genomic sequences SEQ ID NO: 2220-2233, 2234-2298, 2299-2299, 2300-2303, 2304-2313, 2314-2345, 2346-2350, 2351-2351 or 2352-2370.

[0648] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, when the cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva or cerebrospinal fluid) is identified as being from the bladder epithelial cells of a subject, the method indicates that the subject has abnormal cell death and / or suffers from a disease associated with the cell. In some embodiments, the disease or condition is an injury, inflammation or cancer of the bladder epithelium.

[0649] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., bladder epithelial cells) decreases (e.g., is less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., bladder epithelial cells) increases (e.g., is more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two detections, the subject undergoes treatment, and thus the detection results indicate the treatment effect.

[0650] In one embodiment, a method is also provided for determining the cell type of disease cells (e.g., cancer cells), the primary source of the disease (e.g., cancer cells), or the signal or origin of the disease (e.g., cancer cells). In some embodiments, the primary source of the cancer cells is unknown. In some embodiments, the methods described above include detecting the methylation status of one or more DNA fragments of the cancer cells, and the methylation status can then be used to determine that the cells are bladder epithelial cells, as described above.

[0651] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of the cancer cells. In some embodiments, genetic variations can also be present in the DNA fragments, and thus cell type detection can help associate the genetic variations with cancer. In some embodiments, the genetic variations include mutations. In some embodiments, the genetic variations include deletions or insertions. In some embodiments, the genetic variations constitute microsatellite instability. In some embodiments, the genetic variations constitute loss of heterozygosity. In some embodiments, the genetic variations disrupt or alter gene splicing. In some embodiments, the genetic variations cause frameshifts or generate premature stop codons. Once the primary source of the cancer is identified, the subject can be treated with an appropriate regimen for that cancer type.

[0652] B5. Prostatic Epithelium

[0653] Also as provided in Table A, some genomic locations in prostate epithelial cells are consistently undermethylated or overmethylated compared to all other cell types in humans.

[0654] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from prostatic epithelial cells is provided. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites is located within a human genomic sequence selected from SEQ ID NO: 2371 - 2389, 2390 - 2476, 2477 - 2480, 2481 - 2486 or 2487 - 2495 or selected from SEQ ID NO: 2371 - 2389 or 2390 - 2476, or is within less than 100 bp, 200 bp, 500 bp or 1 kb of the foregoing human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from prostatic epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from prostatic epithelial cells. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from prostatic epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from prostatic epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from prostatic epithelial cells.

[0655] In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites in a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites are located within a human genomic sequence selected from SEQ ID NOs: 2496 - 2500, 2501 - 2501 or 2502 - 2520, or within 100 bp, 200 bp, 500 bp or 1 kb of the foregoing human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from prostate epithelial cells. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from prostate epithelial cells. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from prostate epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from prostate epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from prostate epithelial cells.

[0656] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by genomic sequences SEQ ID NOs: 2371 - 2389, 2390 - 2476, 2477 - 2480, 2481 - 2486, 2487 - 2495, 2496 - 2500, 2501 - 2501 or 2502 - 2520.

[0657] The cell type identification method can be used to detect diseases or conditions associated with a cell type. In one embodiment, when cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva or cerebrospinal fluid) is identified as being from prostate epithelial cells of a subject, the method indicates that the subject has abnormal cell death and / or has a disease associated with that cell. In some embodiments, the disease or condition is an injury, inflammation or cancer of the prostate epithelium.

[0658] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., prostate epithelial cells) decreases (e.g., is less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., prostate epithelial cells) increases (e.g., is more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two tests, the subject undergoes treatment, so the test results indicate the treatment effect.

[0659] In one embodiment, a method is also provided for determining the cell type of disease cells (e.g., cancer cells), the primary origin of the disease (e.g., cancer cells), or the signal or source of the disease (e.g., cancer cells). In some embodiments, the primary origin of the cancer cells is unknown. In some embodiments, the methods described above include detecting the methylation status of one or more DNA fragments of the cancer cells, and the methylation status can then be used to determine that the cells are prostate epithelial cells, as described above.

[0660] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of the cancer cells. In some embodiments, genetic variations can also be present in the DNA fragments, so cell type detection can help associate the genetic variations with cancer. In some embodiments, the genetic variations include mutations. In some embodiments, the genetic variations include deletions or insertions. In some embodiments, the genetic variations constitute microsatellite instability. In some embodiments, the genetic variations constitute loss of heterozygosity. In some embodiments, the genetic variations disrupt or alter gene splicing. In some embodiments, the genetic variations cause frameshifts or generate premature stop codons. Once the primary origin of the cancer is identified, the subject can be treated with an appropriate regimen for that cancer type.

[0661] B6. Mammary Basal Epithelium

[0662] Also as provided in Table A, some genomic locations are consistently hypomethylated or hypermethylated in mammary basal epithelial cells compared to all other cell types in humans.

[0663] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from mammary basal epithelial cells is provided. In some embodiments, the method requires detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites in a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites is located within a human genomic sequence selected from SEQ ID NO: 2521 - 2536, 2537 - 2616, 2617 - 2625 or 2626 - 2651 or selected from SEQ ID NO: 2521 - 2536 or 2537 - 2616, or is within less than 100 bp, 200 bp, 500 bp or 1 kb of the foregoing human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from mammary basal epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from mammary basal epithelial cells. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from mammary basal epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from mammary basal epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from mammary basal epithelial cells.

[0664] In some embodiments, the method requires detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites are located within a human genomic sequence selected from SEQ ID NO: 2652 - 2659 or 2660 - 2676 or within a human genomic sequence selected from SEQ ID NO: 2652 - 2659, or within 100 bp, 200 bp, 500 bp or 1 kb of the foregoing human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method subsequently identifies that the target DNA fragment is from mammary basal epithelial cells. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from mammary basal epithelial cells. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from mammary basal epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from mammary basal epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from mammary basal epithelial cells.

[0665] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine the cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by the genomic sequences SEQ ID NO: 2521 - 2536, 2537 - 2616, 2617 - 2625, 2626 - 2651, 2652 - 2659 or 2660 - 2676.

[0666] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, when the cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva or cerebrospinal fluid) is identified as being from the mammary basal epithelial cells of a subject, the method indicates that the subject has abnormal cell death and / or suffers from a disease associated with the cell. In some embodiments, the disease or condition is an injury, inflammation or cancer of the mammary basal epithelium.

[0667] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., mammary basal epithelial cells) decreases (e.g., is less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., mammary basal epithelial cells) increases (e.g., is more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two detections, the subject undergoes treatment, so the detection results indicate the treatment effect.

[0668] In one embodiment, a method for determining the cell type of disease cells (e.g., cancer cells), the primary origin of the disease (e.g., cancer cells), or the signal or origin of the disease (e.g., cancer cells) is also provided. In some embodiments, the primary origin of the cancer cells is unknown. In some embodiments, the methods described above include detecting the methylation status of one or more DNA fragments of the cancer cells, and the methylation status can then be used to determine that the cells are mammary basal epithelial cells, as described above.

[0669] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of the cancer cells. In some embodiments, genetic variations can also be present in the DNA fragments, so cell type detection can help associate the genetic variations with cancer. In some embodiments, the genetic variations include mutations. In some embodiments, the genetic variations include deletions or insertions. In some embodiments, the genetic variations constitute microsatellite instability. In some embodiments, the genetic variations constitute loss of heterozygosity. In some embodiments, the genetic variations disrupt or alter gene splicing. In some embodiments, the genetic variations cause frameshifts or generate premature stop codons. Once the primary origin of the cancer is identified, the subject can be treated with an appropriate regimen for that cancer type.

[0670] B7. Mammary Luminal Epithelium

[0671] Also as provided in Table A, some genomic locations in mammary luminal epithelial cells are consistently undermethylated or overmethylated compared to all other cell types in humans.

[0672] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from mammary luminal epithelial cells is provided. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more) CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten, or all) of the CpG sites is located within a human genomic sequence selected from SEQ ID NO: 2677-2688, 2689-2748, 2749-2749, 2750-2762, or 2763-2802, or is within less than 100 bp, 200 bp, 500 bp, or 1 kb of the foregoing human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from mammary luminal epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from mammary luminal epithelial cells. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from mammary luminal epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from mammary luminal epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from mammary luminal epithelial cells.

[0673] In some embodiments, the method requires detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) CpG sites are located within a human genomic sequence selected from SEQ ID NO: 2803-2815, 2816-2816 or 2817-2827 or selected from SEQ ID NO: 2803-2815, or within 100 bp, 200 bp, 500 bp or 1 kb of the foregoing human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method subsequently identifies that the target DNA fragment is from luminal epithelial cells of the breast. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from luminal epithelial cells of the breast. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from luminal epithelial cells of the breast. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from luminal epithelial cells of the breast. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from luminal epithelial cells of the breast.

[0674] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by genomic sequences SEQ ID NO: 2677-2688, 2689-2748, 2749-2749, 2750-2762, 2763-2802, 2803-2815, 2816-2816 or 2817-2827.

[0675] The cell type identification method can be used to detect diseases or conditions associated with cell type. In one embodiment, when cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva or cerebrospinal fluid) is identified as being from luminal epithelial cells of the breast of a subject, the method indicates that the subject has abnormal cell death and / or has a disease associated with that cell. In some embodiments, the disease or condition is an injury, inflammation or cancer of the luminal epithelium of the breast.

[0676] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., mammary luminal epithelial cells) decreases (e.g., is less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., mammary luminal epithelial cells) increases (e.g., is more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two tests, the subject undergoes treatment, and thus the test results indicate the treatment effect.

[0677] In one embodiment, a method for determining the cell type of disease cells (e.g., cancer cells), the primary origin of the disease (e.g., cancer cells), or the signal or source of the disease (e.g., cancer cells) is also provided. In some embodiments, the primary origin of the cancer cells is unknown. In some embodiments, the methods described above include detecting the methylation status of one or more DNA fragments of the cancer cells, and the methylation status can then be used to determine that the cells are mammary luminal epithelial cells, as described above.

[0678] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of the cancer cells. In some embodiments, genetic variations can also be present in the DNA fragments, and thus cell type detection can help associate the genetic variations with cancer. In some embodiments, the genetic variations include mutations. In some embodiments, the genetic variations include deletions or insertions. In some embodiments, the genetic variations constitute microsatellite instability. In some embodiments, the genetic variations constitute loss of heterozygosity. In some embodiments, the genetic variations disrupt or alter gene splicing. In some embodiments, the genetic variations cause frameshifts or generate premature stop codons. Once the primary origin of the cancer is identified, the subject can be treated with an appropriate regimen for that cancer type.

[0679] Group 6 - Mammary Basal Epithelium and Mammary Luminal Epithelium

[0680] Also as provided in Table A, compared to all other cell types in humans, a group of cells, namely mammary basal epithelial cells, are hypomethylated or hypermethylated at some genomic locations consistently with mammary luminal epithelial cells.

[0681] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from cells selected from mammary basal epithelial cells and mammary luminal epithelial cells is provided. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more) CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten, or all) CpG sites are located within a human genomic sequence selected from SEQ ID NO: 6076 - 6090, 6091 - 6159, 6160 - 6160, 6161 - 6162, 6163 - 6171, or 6172 - 6201, or selected from SEQ ID NO: 6076 - 6090 or 6091 - 6159, or within 100 bp, 200 bp, 500 bp, or 1 kb of the foregoing human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from cells selected from mammary basal epithelial cells and mammary luminal epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from cells selected from mammary basal epithelial cells and mammary luminal epithelial cells. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from cells selected from mammary basal epithelial cells and mammary luminal epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from cells selected from mammary basal epithelial cells and mammary luminal epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from cells selected from mammary basal epithelial cells and mammary luminal epithelial cells.

[0682] In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites in a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites are located within a human genomic sequence selected from SEQ ID NO: 6202 - 6206 or 6207 - 6226 or within a human genomic sequence selected from SEQ ID NO: 6202 - 6206, or are less than 100 bp, 200 bp, 500 bp or 1 kb away from the aforementioned human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from a cell selected from mammary basal epithelial cells and mammary luminal epithelial cells. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from a cell selected from mammary basal epithelial cells and mammary luminal epithelial cells. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from a cell selected from mammary basal epithelial cells and mammary luminal epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from a cell selected from mammary basal epithelial cells and mammary luminal epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from a cell selected from mammary basal epithelial cells and mammary luminal epithelial cells.

[0683] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by the genomic sequences SEQ ID NO: 6076 - 6090, 6091 - 6159, 6160 - 6160, 6161 - 6162, 6163 - 6171, 6172 - 6201, 6202 - 6206 or 6207 - 6226.

[0684] A method for cell type identification can be used to detect diseases or conditions associated with cell types. In one embodiment, when cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva, or cerebrospinal fluid) is identified as being from cells selected from the mammary basal epithelium and the mammary luminal epithelium of a subject, the method indicates that the subject has abnormal cell death and / or is suffering from a disease associated with such cells. In some embodiments, the disease or condition is injury, inflammation, or cancer of cells selected from the mammary basal epithelium and the mammary luminal epithelium.

[0685] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., cells selected from the mammary basal epithelium and the mammary luminal epithelium) decreases (e.g., is less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the above method includes making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., cells selected from the mammary basal epithelium and the mammary luminal epithelium) increases (e.g., is more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the above method includes making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two detections, the subject undergoes treatment, and thus the detection results indicate the treatment effect.

[0686] In one embodiment, a method for determining the cell type of diseased cells (e.g., cancer cells), the primary origin of a disease (e.g., cancer cells), or the signal or source of a disease (e.g., cancer cells) is also provided. In some embodiments, the primary origin of the cancer cells is unknown. In some embodiments, the above method includes detecting the methylation status of one or more DNA fragments of the cancer cells, and then the methylation status can be used to determine that the cells are cells selected from the mammary basal epithelium and the mammary luminal epithelium, as described above.

[0687] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of the cancer cells. In some embodiments, genetic variations can also be present in the DNA fragments, and thus cell type detection can help to correlate the genetic variation with cancer. In some embodiments, the genetic variation includes a mutation. In some embodiments, the genetic variation includes a deletion or an insertion. In some embodiments, the genetic variation constitutes microsatellite instability. In some embodiments, the genetic variation constitutes loss of heterozygosity. In some embodiments, the genetic variation disrupts or alters gene splicing. In some embodiments, the genetic variation causes a frameshift or generates a premature stop codon. Once the primary source of the cancer has been identified, the subject can be treated with an appropriate regimen for that cancer type.

[0688] Group 7 - Oviductal Epithelium, Ovarian Epithelium, and Endometrial Epithelium

[0689] Also as provided in Table A, compared to all other cell types in humans, a group of cells, namely fallopian tube epithelium, ovarian epithelium, and some genomic locations in endometrial epithelium, are hypomethylated or hypermethylated consistently.

[0690] According to one embodiment of the present disclosure, there is provided a method for identifying that a biological sample contains DNA from cells selected from fallopian tube epithelium, ovarian epithelium, and endometrial epithelium. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more) CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten, or all) CpG sites are located within a human genomic sequence selected from SEQ ID NOs: 6366 - 6399, 6400 - 6468, 6469 - 6475, 6476 - 6491, or 6492 - 6515, or within a human genomic sequence selected from SEQ ID NOs: 6366 - 6399 or 6400 - 6468, or within 100 bp, 200 bp, 500 bp, or 1 kb of the foregoing human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from cells selected from fallopian tube epithelium, ovarian epithelium, and endometrial epithelium. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from cells selected from fallopian tube epithelium, ovarian epithelium, and endometrial epithelium. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from cells selected from fallopian tube epithelium, ovarian epithelium, and endometrial epithelium. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from cells selected from fallopian tube epithelium, ovarian epithelium, and endometrial epithelium. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from cells selected from fallopian tube epithelium, ovarian epithelium, and endometrial epithelium.

[0691] In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites in a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites are located within the human genomic sequence selected from SEQ ID NO: 6516 - 6527 or 6528 - 6540 or within the human genomic sequence selected from SEQ ID NO: 6516 - 6527, or are less than 100 bp, 200 bp, 500 bp or 1 kb away from the aforementioned human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from cells selected from fallopian tube epithelium, ovarian epithelium, and endometrial epithelium. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from cells selected from fallopian tube epithelium, ovarian epithelium, and endometrial epithelium. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from cells selected from fallopian tube epithelium, ovarian epithelium, and endometrial epithelium. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from cells selected from fallopian tube epithelium, ovarian epithelium, and endometrial epithelium. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from cells selected from fallopian tube epithelium, ovarian epithelium, and endometrial epithelium.

[0692] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine the cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by the genomic sequences SEQ ID NO: 6366 - 6399, 6400 - 6468, 6469 - 6475, 6476 - 6491, 6492 - 6515, 6516 - 6527 or 6528 - 6540.

[0693] A method for cell type identification can be used to detect diseases or conditions associated with cell types. In one embodiment, when cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva, or cerebrospinal fluid) is identified as being from cells selected from fallopian tube epithelium, ovarian epithelium, and endometrial epithelium of a subject, the method indicates that the subject has abnormal cell death and / or is suffering from a disease associated with such cells. In some embodiments, the disease or condition is injury, inflammation, or cancer of cells selected from fallopian tube epithelium, ovarian epithelium, and endometrial epithelium.

[0694] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., cells selected from fallopian tube epithelium, ovarian epithelium, and endometrial epithelium) is decreased (e.g., less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the method described above includes making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., cells selected from fallopian tube epithelium, ovarian epithelium, and endometrial epithelium) is increased (e.g., more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the method described above includes making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two detections, the subject undergoes treatment, and thus the detection results indicate the treatment effect.

[0695] In one embodiment, a method for determining the cell type of disease cells (e.g., cancer cells), the primary source of a disease (e.g., cancer cells), or the signal or origin of a disease (e.g., cancer cells) is also provided. In some embodiments, the primary source of the cancer cells is unknown. In some embodiments, the method described above includes detecting the methylation status of one or more DNA fragments of the cancer cells, and then the methylation status can be used to determine that the cells are cells selected from fallopian tube epithelium, ovarian epithelium, and endometrial epithelium, as described above.

[0696] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus cell type detection can help to correlate the genetic variation with cancer. In some embodiments, the genetic variation includes a mutation. In some embodiments, the genetic variation includes a deletion or an insertion. In some embodiments, the genetic variation constitutes microsatellite instability. In some embodiments, the genetic variation constitutes loss of heterozygosity. In some embodiments, the genetic variation disrupts or alters gene splicing. In some embodiments, the genetic variation causes a frameshift or generates a premature stop codon. Once the primary source of the cancer has been identified, the subject can be treated with an appropriate regimen for that cancer type.

[0697] C. Cardiovascular - Lung Cells

[0698] C1. Alveolar Epithelium

[0699] Also as provided in Table A, some genomic locations in alveolar epithelial cells are consistently hypomethylated or hypermethylated compared to all other cell types in humans.

[0700] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from alveolar epithelial cells is provided. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites is located within a human genomic sequence selected from SEQ ID NO: 2828 - 2838, 2839 - 2899, 2900 - 2900, 2901 - 2903, 2904 - 2916 or 2917 - 2953, or is less than 100 bp, 200 bp, 500 bp or 1 kb away from the aforementioned human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from alveolar epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from alveolar epithelial cells. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from alveolar epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from alveolar epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from alveolar epithelial cells.

[0701] In some embodiments, the method requires detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites are located within the human genomic sequence selected from SEQ ID NO: 2954-2960 or 2961-2978 or selected from SEQ ID NO: 2954-2960, or are less than 100 bp, 200 bp, 500 bp or 1 kb away from the aforementioned human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from alveolar epithelial cells. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from alveolar epithelial cells. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from alveolar epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from alveolar epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from alveolar epithelial cells.

[0702] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by the genomic sequences SEQ ID NO: 2828-2838, 2839-2899, 2900-2900, 2901-2903, 2904-2916, 2917-2953, 2954-2960 or 2961-2978.

[0703] Example 2 of the present disclosure discloses a set of methylation markers capable of distinguishing different lung cell types, such as alveolar cells or bronchial cells. Example markers are provided in Table 3. Seventeen genomic loci are uniquely unmethylated or hypermethylated in lung epithelial cells, including three loci specifically identifying bronchial cells, twelve loci specifically identifying alveolar cells, and two loci that can identify both cell types. Using the reference chromosomal positions as a reference, the two loci identifying both bronchial cells and alveolar cells are chromosome 14:55765534 (hg19, the same below; reference gene: FBXO34) and chromosome 3:181441571 (reference gene: SOX2OT); the twelve loci specifically identifying alveolar cells are chromosome 1:41486102 (reference gene: SLFNL1), chromosome 2:236672684 (reference gene: AGAP1), chromosome 17:79952367 (reference gene: ASPSCR1), chromosome 16:678127 (reference gene: RAB40C), chromosome 7:2473529 (reference gene: CHST12), chromosome 16:1652552 (reference gene: IFT140), chromosome 14:91691190 (reference gene: C14orf159), chromosome 16:667157 (reference gene: RAB40C), chromosome 11:66116455 (reference gene: B3GNTl), chromosome 4:57522145 (reference gene: HOPX), chromosome 16:84271391 (reference gene: KCNG4), and chromosome 1:1986275 (reference gene: PRKCZ); the three loci specifically identifying bronchial cells are chromosome 7:4802132 (reference gene: FOXK1), chromosome 2:239970075 (reference gene: HDAC4), and chromosome 1:164761834 (reference gene: PBX1).

[0704] For example, as Figure 9 shown, the genomic marker sequence at the Rab40C gene is only unmethylated in alveolar epithelium, but not in bronchial cells. As Figure 13 demonstrated, when using the methylation status of one or more of these markers, lung cell types can be easily distinguished. When using the first three markers, the performance is close to that when using all seventeen markers, highlighting the robustness of the technique.

[0705] Accordingly, in one embodiment, provided is a method for identifying that a biological sample contains DNA from lung cells, the method comprising detecting the methylation status of each of at least four CpG sites of a target DNA fragment in the biological sample; if the methylation status corresponds to that of reference human alveolar cells or human lung bronchial cells, wherein according to the human genome assembly version hg19, the target DNA fragment is less than 1 kb away from a genomic locus selected from the group consisting of the following human chromosomes: chromosome 14:55765534, chromosome 3:181441571, chromosome 1:41486102, chromosome 2:236672684, chromosome 17:79952367, chromosome 16:678127, chromosome 7:2473529, chromosome 16:1652552, chromosome 14:91691190, chromosome 16:667157, chromosome 11:66116455, chromosome 4:57522145, chromosome 16:84271391, chromosome 1:1986275, chromosome 7:4802132, chromosome 2:239970075, chromosome 1:164761834, then the target DNA fragment is identified as being from human alveolar cells or human lung bronchial cells.

[0706] As used herein, in some embodiments, the methylation status refers to the percentage of CpG sites within a genomic sequence that are methylated. In some embodiments, the methylation status simply refers to hypermethylation (M, at least 60% CpG methylation) or hypomethylation (U, no more than 40% CpG methylation).

[0707] For example, in one embodiment, if the target DNA fragment is unmethylated and close to a genomic locus of chromosome 2:236672684, chromosome 17:79952367, chromosome 16:678127, chromosome 7:2473529, chromosome 16:1652552, chromosome 14:91691190, chromosome 16:667157, chromosome 11:66116455, chromosome 16:84271391 or chromosome 1:1986275, then the target DNA fragment is identified as being from human alveolar cells. In one embodiment, if the target DNA fragment is methylated and close to a genomic locus of chromosome 4:57522145, then the target DNA fragment is identified as being from human alveolar cells.

[0708] In one embodiment, if the target DNA fragment is unmethylated and close to a genomic locus of chromosome 7:4802132, chromosome 2:239970075 or chromosome 1:164761834, then the target DNA fragment is identified as being from human lung bronchial cells.

[0709] In one embodiment, if the target DNA fragment is unmethylated and close to the genomic locus of chromosome 14:55765534 or chromosome 1:41486102, or methylated and close to the genomic locus of chromosome 3:181441571, then the target DNA fragment is identified as a human alveolar cell or a human lung bronchial cell.

[0710] In some embodiments, the DNA fragment containing the CpG site for measurement is less than 1000 bp away from a reference genomic location (e.g., chromosome 14:55765534). In some embodiments, the DNA fragment containing the CpG site for measurement is less than 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 400 bp, 300 bp, 250 bp, 200 bp, or 150 bp away from the reference genomic location.

[0711] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, when cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva, or cerebrospinal fluid) is identified as being from the alveolar epithelial cells of a subject, the method indicates that the subject has abnormal cell death and / or suffers from a disease associated with the cell. In some embodiments, the disease or condition is damage, inflammation, or cancer of the alveolar epithelium.

[0712] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a specific type (e.g., alveolar epithelial cells) decreases (e.g., less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the above method includes making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a specific type (e.g., alveolar epithelial cells) increases (e.g., more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the above method includes making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two detections, the subject undergoes treatment, so the detection results indicate the treatment effect.

[0713] In one embodiment, a method is also provided for determining the cell type of a diseased cell (e.g., a cancer cell), the primary origin of a disease (e.g., a cancer cell), or the signal or origin of a disease (e.g., a cancer cell). In some embodiments, the primary origin of the cancer cell is unknown. In some embodiments, the method includes detecting the methylation status of one or more DNA fragments of the cancer cell, and the methylation status can then be used to determine that the cell is an alveolar epithelial cell, as described above.

[0714] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of the cancer cell. In some embodiments, genetic variations may also be present in the DNA fragment, and thus cell type detection can help associate the genetic variation with cancer. In some embodiments, the genetic variation includes a mutation. In some embodiments, the genetic variation includes a deletion or an insertion. In some embodiments, the genetic variation constitutes microsatellite instability. In some embodiments, the genetic variation constitutes loss of heterozygosity. In some embodiments, the genetic variation disrupts or alters gene splicing. In some embodiments, the genetic variation causes a frameshift or generates a premature stop codon. Once the primary origin of the cancer is identified, the subject can be treated with an appropriate regimen for that cancer type.

[0715] C2. Pulmonary Bronchial Epithelium

[0716] Also as provided in Table A, some genomic locations in lung bronchial epithelial cells are consistently hypomethylated or hypermethylated compared to all other cell types in humans.

[0717] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from lung bronchial epithelial cells is provided. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites is located within a human genomic sequence selected from SEQ ID NO: 2979 - 3001, 3002 - 3087, 3088 - 3090, 3091 - 3092 or 3093 - 3104 or selected from SEQ ID NO: 2979 - 3001 or 3002 - 3087, or is less than 100 bp, 200 bp, 500 bp or 1 kb away from the foregoing human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from lung bronchial epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from lung bronchial epithelial cells. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from lung bronchial epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from lung bronchial epithelial cells.

[0718] In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from lung bronchial epithelial cells.

[0719] In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites is located within a human genomic sequence selected from SEQ ID NOs: 3105 - 3109 or 3110 - 3129 or within a human genomic sequence selected from SEQ ID NOs: 3105 - 3109, or is less than 100 bp, 200 bp, 500 bp or 1 kb away from the aforementioned human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from lung bronchial epithelial cells. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from lung bronchial epithelial cells. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from lung bronchial epithelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from lung bronchial epithelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from lung bronchial epithelial cells.

[0720] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by the genomic sequences SEQ ID NOs: 2979 - 3001, 3002 - 3087, 3088 - 3090, 3091 - 3092, 3093 - 3104, 3105 - 3109 or 3110 - 3129.

[0721] The cell type identification method can be used to detect diseases or conditions associated with cell type. In one embodiment, when cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva or cerebrospinal fluid) is identified as being from the lung bronchial epithelial cells of a subject, the method indicates that the subject has abnormal cell death and / or has a disease associated with that cell. In some embodiments, the disease or condition is injury, inflammation or cancer of the lung bronchial epithelium.

[0722] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., lung bronchial epithelial cells) decreases (e.g., is less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., lung bronchial epithelial cells) increases (e.g., is more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two detections, the subject undergoes treatment, and thus the detection results indicate the treatment effect.

[0723] In one embodiment, a method for determining the cell type of disease cells (e.g., cancer cells), the primary source of the disease (e.g., cancer cells), or the signal or origin of the disease (e.g., cancer cells) is also provided. In some embodiments, the primary source of the cancer cells is unknown. In some embodiments, the methods described above include detecting the methylation status of one or more DNA fragments of the cancer cells, and the methylation status can then be used to determine that the cells are lung bronchial epithelial cells, as described above.

[0724] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of the cancer cells. In some embodiments, genetic variations can also be present in the DNA fragments, and thus cell type detection can help associate the genetic variations with cancer. In some embodiments, the genetic variations include mutations. In some embodiments, the genetic variations include deletions or insertions. In some embodiments, the genetic variations constitute microsatellite instability. In some embodiments, the genetic variations constitute loss of heterozygosity. In some embodiments, the genetic variations disrupt or alter gene splicing. In some embodiments, the genetic variations cause a frameshift or generate a premature stop codon. Once the primary source of the cancer is identified, the subject can be treated with an appropriate regimen for that cancer type.

[0725] C3. Cardiac Myocytes

[0726] Also as provided in Table A, some genomic locations in heart cardiomyocytes are consistently hypomethylated or hypermethylated compared to all other cell types in humans.

[0727] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from cardiac cardiomyocytes is provided. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites is located within a human genomic sequence selected from SEQ ID NO: 3130 - 3147, 3148 - 3223, 3224 - 3230 or 3231 - 3254 or selected from SEQ ID NO: 3130 - 3147 or 3148 - 3223, or is less than 100 bp, 200 bp, 500 bp or 1 kb away from the foregoing human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from cardiac cardiomyocytes. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from cardiac cardiomyocytes. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from cardiac cardiomyocytes. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from cardiac cardiomyocytes. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from cardiac cardiomyocytes.

[0728] In some embodiments, the method requires detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) CpG sites are located within a human genomic sequence selected from SEQ ID NO: 3255 - 3266, 3267 - 3267 or 3268 - 3279 or within a human genomic sequence selected from SEQ ID NO: 3255 - 3266, or within 100 bp, 200 bp, 500 bp or 1 kb of the foregoing human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method subsequently identifies that the target DNA fragment is from cardiac cardiomyocytes. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from cardiac cardiomyocytes. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from cardiac cardiomyocytes. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from cardiac cardiomyocytes. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from cardiac cardiomyocytes.

[0729] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine the cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by the genomic sequences SEQ ID NO: 3130 - 3147, 3148 - 3223, 3224 - 3230, 3231 - 3254, 3255 - 3266, 3267 - 3267 or 3268 - 3279.

[0730] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, when the cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva or cerebrospinal fluid) is identified as being from the cardiac cardiomyocytes of a subject, the method indicates that the subject has abnormal cell death and / or suffers from a disease associated with the cell. In some embodiments, the disease or condition is damage, inflammation or cancer of the cardiac cardiomyocytes.

[0731] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., cardiac cardiomyocytes) decreases (e.g., is less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition based on the recovery indication accordingly. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., cardiac cardiomyocytes) increases (e.g., is more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition based on the worsening indication accordingly. In some embodiments, between more than two detections, the subject undergoes treatment, so the detection results indicate the treatment effect.

[0732] In one embodiment, a method for determining the cell type of disease cells (e.g., cancer cells), the primary origin of the disease (e.g., cancer cells), or the signal or source of the disease (e.g., cancer cells) is also provided. In some embodiments, the primary origin of the cancer cells is unknown. In some embodiments, the methods described above include detecting the methylation status of one or more DNA fragments of the cancer cells, and the methylation status can then be used to determine that the cells are cardiac cardiomyocytes, as described above.

[0733] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of the cancer cells. In some embodiments, genetic variations can also be present in the DNA fragments, so cell type detection can help associate the genetic variations with cancer. In some embodiments, the genetic variations include mutations. In some embodiments, the genetic variations include deletions or insertions. In some embodiments, the genetic variations constitute microsatellite instability. In some embodiments, the genetic variations constitute loss of heterozygosity. In some embodiments, the genetic variations disrupt or alter gene splicing. In some embodiments, the genetic variations cause frameshifts or generate premature stop codons. Once the primary origin of the cancer is identified, the subject can be treated with an appropriate regimen for that cancer type.

[0734] C4. Cardiac Fibroblasts

[0735] Also as provided in Table A, some genomic locations in cardiac fibroblasts are consistently undermethylated or overmethylated compared to all other cell types in humans.

[0736] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from cardiac fibroblasts is provided. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more) CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten, or all) of the CpG sites is located within a human genomic sequence selected from SEQ ID NO: 33280-3300, 3301-3394, 3395-3396, 3397-3400, or 3401-3407, or selected from SEQ ID NO: 3280-3300 or 3301-3394, or is less than 100 bp, 200 bp, 500 bp, or 1 kb away from the aforementioned human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from cardiac fibroblasts. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from cardiac fibroblasts. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from cardiac fibroblasts. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from cardiac fibroblasts. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from cardiac fibroblasts.

[0737] In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites in a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites are located within a human genomic sequence selected from SEQ ID NOs: 3408 - 3414, 3415 - 3416 or 3417 - 3432 or within a human genomic sequence selected from SEQ ID NOs: 3408 - 3414, or within 100 bp, 200 bp, 500 bp or 1 kb of the foregoing human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from cardiac fibroblasts. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from cardiac fibroblasts. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from cardiac fibroblasts. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from cardiac fibroblasts. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from cardiac fibroblasts.

[0738] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by genomic sequences SEQ ID NOs: 3280 - 3300, 3301 - 3394, 3395 - 3396, 3397 - 3400, 3401 - 3407, 3408 - 3414, 3415 - 3416 or 3417 - 3432.

[0739] The cell type identification method can be used to detect diseases or conditions associated with cell type. In one embodiment, when cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva or cerebrospinal fluid) is identified as being from the cardiac fibroblasts of a subject, the method indicates that the subject has abnormal cell death and / or has a disease associated with that cell. In some embodiments, the disease or condition is injury, inflammation or cancer of cardiac fibroblasts.

[0740] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., cardiac fibroblasts) decreases (e.g., is less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., cardiac fibroblasts) increases (e.g., is more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two tests, the subject undergoes treatment, and thus the test results indicate the treatment effect.

[0741] In one embodiment, a method is also provided for determining the cell type of disease cells (e.g., cancer cells), the primary origin of the disease (e.g., cancer cells), or the signal or origin of the disease (e.g., cancer cells). In some embodiments, the primary origin of the cancer cells is unknown. In some embodiments, the methods described above include detecting the methylation status of one or more DNA fragments of the cancer cells, and the methylation status can then be used to determine that the cells are cardiac fibroblasts, as described above.

[0742] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of the cancer cells. In some embodiments, genetic variations can also be present in the DNA fragments, and thus cell type detection can help associate the genetic variations with cancer. In some embodiments, the genetic variations include mutations. In some embodiments, the genetic variations include deletions or insertions. In some embodiments, the genetic variations constitute microsatellite instability. In some embodiments, the genetic variations constitute loss of heterozygosity. In some embodiments, the genetic variations disrupt or alter gene splicing. In some embodiments, the genetic variations cause frameshifts or generate premature stop codons. Once the primary origin of the cancer is identified, the subject can be treated with an appropriate regimen for that cancer type.

[0743] C5. Vascular Endothelial Cells

[0744] Also as provided in Table A, some genomic locations are consistently hypomethylated or hypermethylated in vascular endothelial cells compared to all other cell types in humans.

[0745] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from vascular endothelial cells is provided. In some embodiments, the method requires detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites is located within a human genomic sequence selected from SEQ ID NO: 3433 - 3456, 3457 - 3547, 3548 - 3550, 3551 - 3551 or 3552 - 3559 or selected from SEQ ID NO: 3433 - 3456 or 3457 - 3547, or is less than 100 bp, 200 bp, 500 bp or 1 kb away from the aforementioned human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from vascular endothelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from vascular endothelial cells. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from vascular endothelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from vascular endothelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from vascular endothelial cells.

[0746] In some embodiments, the method requires detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) CpG sites are located within a human genomic sequence selected from SEQ ID NO: 3560 - 3579, 3580 - 3580 or 3581 - 3584 or within a human genomic sequence selected from SEQ ID NO: 3560 - 3579, or within 100 bp, 200 bp, 500 bp or 1 kb of the foregoing human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from vascular endothelial cells. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from vascular endothelial cells. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from vascular endothelial cells. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from vascular endothelial cells. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from vascular endothelial cells.

[0747] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by the genomic sequences SEQ ID NO: 3433 - 3456, 3457 - 3547, 3548 - 3550, 3551 - 3551, 3552 - 3559, 3560 - 3579, 3580 - 3580 or 3581 - 3584.

[0748] The cell type identification method can be used to detect diseases or conditions associated with cell type. In one embodiment, when cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva or cerebrospinal fluid) is identified as being from the vascular endothelial cells of a subject, the method indicates that the subject has abnormal cell death and / or has a disease associated with that cell. In some embodiments, the disease or condition is injury, inflammation or cancer of vascular endothelial cells.

[0749] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., vascular endothelial cells) decreases (e.g., less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., vascular endothelial cells) increases (e.g., more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two detections, the subject undergoes treatment, and thus the detection results indicate the treatment effect.

[0750] In one embodiment, a method for determining the cell type of disease cells (e.g., cancer cells), the primary origin of the disease (e.g., cancer cells), or the signal or source of the disease (e.g., cancer cells) is also provided. In some embodiments, the primary origin of the cancer cells is unknown. In some embodiments, the methods described above include detecting the methylation status of one or more DNA fragments of the cancer cells, and the methylation status can then be used to determine that the cells are vascular endothelial cells, as described above.

[0751] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of the cancer cells. In some embodiments, genetic variations can also be present in the DNA fragments, and thus cell type detection can help associate the genetic variations with cancer. In some embodiments, the genetic variations include mutations. In some embodiments, the genetic variations include deletions or insertions. In some embodiments, the genetic variations constitute microsatellite instability. In some embodiments, the genetic variations constitute loss of heterozygosity. In some embodiments, the genetic variations disrupt or alter gene splicing. In some embodiments, the genetic variations cause frameshifts or generate premature stop codons. Once the primary origin of the cancer is identified, the subject can be treated with an appropriate regimen for that cancer type.

[0752] Group 8 - Cardiac Myocytes and Cardiac Fibroblasts

[0753] Also as provided in Table A, compared to all other cell types in humans, a group of cells, namely cardiac cardiomyocytes and some of the cardiac fibroblasts, are hypomethylated or hypermethylated at some genomic locations in a consistent manner.

[0754] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from cells selected from cardiac myocytes and cardiac fibroblasts is provided. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites is located within a human genomic sequence selected from SEQ ID NO: 6940 - 6959, 6960 - 7045, 7046 - 7046, 7047 - 7049, 7050 - 7053 or 7054 - 7065 or selected from SEQ ID NO: 6940 - 6959 or 6960 - 7045, or is within less than 100 bp, 200 bp, 500 bp or 1 kb of the foregoing human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from cells selected from cardiac myocytes and cardiac fibroblasts. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from cells selected from cardiac myocytes and cardiac fibroblasts. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from cells selected from cardiac myocytes and cardiac fibroblasts. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from cells selected from cardiac myocytes and cardiac fibroblasts. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from cells selected from cardiac myocytes and cardiac fibroblasts.

[0755] In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites in a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites are located within a human genomic sequence selected from SEQ ID NO: 7066 - 7082 or 7083 - 7090 or within a human genomic sequence selected from SEQ ID NO: 7066 - 7082, or within 100 bp, 200 bp, 500 bp or 1 kb of the aforementioned human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from a cell selected from cardiac cardiomyocytes and cardiac fibroblasts. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from a cell selected from cardiac cardiomyocytes and cardiac fibroblasts. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from a cell selected from cardiac cardiomyocytes and cardiac fibroblasts. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from a cell selected from cardiac cardiomyocytes and cardiac fibroblasts. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from a cell selected from cardiac cardiomyocytes and cardiac fibroblasts.

[0756] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by the genomic sequences SEQ ID NO: 6940 - 6959, 6960 - 7045, 7046 - 7046, 7047 - 7049, 7050 - 7053, 7054 - 7065, 7066 - 7082 or 7083 - 7090.

[0757] Cell type identification methods can be used to detect diseases or conditions associated with cell types. In one embodiment, when cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva, or cerebrospinal fluid) is identified as being from cells selected from a subject's cardiac cardiomyocytes and cardiac fibroblasts, the method indicates that the subject has abnormal cell death and / or is suffering from a disease associated with that cell. In some embodiments, the disease or condition is injury, inflammation, or cancer of a cell selected from cardiac cardiomyocytes and cardiac fibroblasts.

[0758] In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., cells selected from cardiac cardiomyocytes and cardiac fibroblasts) is decreased (e.g., less at a second measurement time point than at an earlier first measurement time point), it indicates that the subject is recovering from the disease or condition. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the recovery indication. In some embodiments, when the amount of cell-free DNA identified as being from one or more cells of a particular type (e.g., cells selected from cardiac cardiomyocytes and cardiac fibroblasts) is increased (e.g., more at a second measurement time point than at an earlier first measurement time point), it indicates that the disease or condition is worsening. In some versions, the methods described above include making a diagnosis and / or treating the disease or condition accordingly based on the worsening indication. In some embodiments, between more than two detections, the subject undergoes treatment, such that the detection results indicate the treatment effect.

[0759] In one embodiment, a method is also provided for determining the cell type of disease cells (e.g., cancer cells), the primary source of a disease (e.g., cancer cells), or the signal or origin of a disease (e.g., cancer cells). In some embodiments, the primary source of the cancer cells is unknown. In some embodiments, the methods described above include detecting the methylation status of one or more DNA fragments of the cancer cells, and the methylation status can then be used to determine that the cells are cells selected from cardiac cardiomyocytes and cardiac fibroblasts, as described above.

[0760] In some cases, cell-free DNA fragments are released from cancer cells. The techniques of the present invention can include determining the cell type of the cancer cells. In some embodiments, genetic variations can also be present in the DNA fragments, and thus cell type detection can help associate the genetic variations with cancer. In some embodiments, the genetic variations include mutations. In some embodiments, the genetic variations include deletions or insertions. In some embodiments, the genetic variations constitute microsatellite instability. In some embodiments, the genetic variations constitute loss of heterozygosity. In some embodiments, the genetic variations disrupt or alter gene splicing. In some embodiments, the genetic variations cause frameshifts or generate premature stop codons. Once the primary source of the cancer is identified, the subject can be treated with an appropriate regimen for that cancer type.

[0761] Group 9 - Alveolar Epithelium and Pulmonary Bronchial Epithelium

[0762] Also as provided in Table A, compared to all other cell types in humans, a set of cells, namely alveolar epithelium and some genomic locations in lung bronchiolar epithelium, are consistently hypomethylated or hypermethylated.

[0763] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from cells selected from alveolar epithelium and lung bronchial epithelium is provided. In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites is located within a human genomic sequence selected from SEQ ID NO: 6227-6243, 6244-6326, 6327-6327, 6328-6329, 6330-6336 or 6337-6352 or selected from SEQ ID NO: 6227-6243 or 6244-6326, or is less than 100 bp, 200 bp, 500 bp or 1 kb away from the foregoing human genomic sequence. In some embodiments, when no more than 40% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from cells selected from alveolar epithelium and lung bronchial epithelium. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is from cells selected from alveolar epithelium and lung bronchial epithelium. Similarly, in some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from cells selected from alveolar epithelium and lung bronchial epithelium. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from cells selected from alveolar epithelium and lung bronchial epithelium. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from cells selected from alveolar epithelium and lung bronchial epithelium.

[0764] In some embodiments, the method involves detecting the methylation status of multiple (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG sites of a target DNA fragment in a biological sample, wherein at least one (or at least two, three, four, five, six, seven, eight, nine, ten or all) of the CpG sites are within the human genomic sequence selected from SEQ ID NO: 6353 or 6354 - 6365, or within 100 bp, 200 bp, 500 bp or 1 kb of the aforementioned human genomic sequence. In some embodiments, when more than 50% of the CpG sites are methylated, the method then identifies that the target DNA fragment is from a cell selected from alveolar epithelium and lung bronchial epithelium. In some embodiments, when at least 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are methylated, the method identifies that the target DNA fragment is from a cell selected from alveolar epithelium and lung bronchial epithelium. Similarly, in some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is from a cell selected from alveolar epithelium and lung bronchial epithelium. In some embodiments, when no more than 25%, 30%, 35%, 40%, 45% or 50% of the CpG sites are methylated, the method identifies that the target DNA fragment is not from a cell selected from alveolar epithelium and lung bronchial epithelium. In some embodiments, when at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the CpG sites are unmethylated, the method identifies that the target DNA fragment is not from a cell selected from alveolar epithelium and lung bronchial epithelium.

[0765] In some embodiments, the methylation status of one or more other DNA fragments is further used to determine the cell type. In some embodiments, one or more additional (different from the first) DNA fragments are represented by the genomic sequences SEQ ID NO: 6227 - 6243, 6244 - 6326, 6327 - 6327, 6328 - 6329, 6330 - 6336, 6337 - 6352, 6353 or 6354 - 6365.

[0766] A method for cell type identification can be used to detect diseases or conditions associated with cell types. In one embodiment, when cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva, or cerebrospinal fluid) is identified as being from cells selected from the alveolar epithelium and lung bronchial epithelium of a subject, the method indicates that the subject has abnormal cell death and / or is suffering from a disease associated with th...

Claims

1. A method for identifying that a biological sample contains DNA from a cell type, characterized in that: the method comprises: detecting the methylation status of each of at least four CpG sites of a target DNA fragment in the biological sample; and identifying the target DNA fragment as: (1) from human oral, laryngeal or esophageal epithelial cells when no more than 40% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 1-90, or when more than 50% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 126-133; (2) from human gastric epithelial cells when no more than 40% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 151-330, or when more than 50% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 379-401; (3) from human small intestinal epithelial cells when no more than 40% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 429-527, or when more than 50% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 555-564; (4) from human colonic epithelial cells when no more than 40% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 580-657, or when more than 50% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 705-715; (5) from human colonic fibroblasts when no more than 40% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 730-732, or when more than 50% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 733-739; (6) When no more than 40% of the CpG sites are methylated, where at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 742 - 829, or when more than 50% of the CpG sites are methylated, where at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 868 - 875, it is from human gallbladder epithelial cells; (7) When no more than 40% of the CpG sites are methylated, where at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 877 - 980, or when more than 50% of the CpG sites are methylated, where at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 1003 - 1018, it is from human liver hepatocytes; (8) When no more than 40% of the CpG sites are methylated, where at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 1028 - 1112, or when more than 50% of the CpG sites are methylated, where at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 1156 - 1161, it is from human pancreatic acinar cells; (9) When no more than 40% of the CpG sites are methylated, where at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 1181 - 1282, or when more than 50% of the CpG sites are methylated, where at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 1307 - 1315, it is from human pancreatic alpha cells; (10) When no more than 40% of the CpG sites are methylated, where at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 1332 - 1440, or when more than 50% of the CpG sites are methylated, where at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 1461 - 1471, it is from human pancreatic beta cells; (11) When no more than 40% of the CpG sites are methylated, where at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 1486 - 1594, or when more than 50% of the CpG sites are methylated, where at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 1614 - 1624, it is from human pancreatic delta cells; or (12) When no more than 40% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 1639 - 1742, or when more than 50% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 1768 - 1779, it is from human pancreatic duct cells.

2. The method according to claim 1, wherein: it further includes detecting the methylation status of each of at least four CpG sites of a second target DNA fragment in the biological sample, and then identifying the second target DNA fragment as: (1’) When no more than 40% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 1 - 125, or when more than 50% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 126 - 150, it is from human oral, laryngeal or esophageal epithelial cells; (2’) When no more than 40% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 151 - 378, or when more than 50% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 379 - 428, it is from human gastric epithelial cells; (3’) When no more than 40% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 429 - 554, or when more than 50% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 555 - 579, it is from human small intestinal epithelial cells; (4’) When no more than 40% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 580 - 704, or when more than 50% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 705 - 729, it is from human colonic epithelial cells; (5’) When no more than 40% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 730 - 732, or when more than 50% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 733 - 741, it is from human colonic fibroblasts; (6’) When no more than 40% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 742 - 867, or when more than 50% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 868 - 876, it is from human gallbladder epithelial cells; (7’) When no more than 40% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 877 - 1002, or when more than 50% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 1003 - 1027, it is from human liver hepatocytes; (8’) When no more than 40% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 1028 - 1155, or when more than 50% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 1156 - 1180, it is from human pancreatic acinar cells; (9’) When no more than 40% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 1181 - 1306, or when more than 50% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 1307 - 1331, it is from human pancreatic alpha cells; (10’) When no more than 40% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 1332 - 1460, or when more than 50% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 1461 - 1485, it is from human pancreatic beta cells; (11’) When no more than 40% of the CpG sites are methylated, with at least one of the CpG sites located within a human genomic sequence selected from the group consisting of SEQ ID NOs: 1486 - 1613, or when more than 50% of the CpG sites are methylated, with at least one of the CpG sites located within a human genomic sequence selected from the group consisting of SEQ ID NOs: 1614 - 1638, it is from human pancreatic delta cells; or (12’) When no more than 40% of the CpG sites are methylated, with at least one of the CpG sites located within a human genomic sequence selected from the group consisting of SEQ ID NOs: 1639 - 1767, or when more than 50% of the CpG sites are methylated, with at least one of the CpG sites located within a human genomic sequence selected from the group consisting of SEQ ID NOs: 1768 - 1792, it is from human pancreatic duct cells.

3. The method according to claim 1 or 2, wherein: the biological sample comprises blood, plasma, serum, semen, milk, urine, saliva or cerebrospinal fluid obtained from a human subject.

4. The method according to claim 3, wherein: the target DNA fragment is a cell-free DNA fragment.

5. The method according to claim 4, wherein: identifying the cell-free DNA fragment as being from a cell type includes detecting abnormal cell death of the cell type, or a disease associated with the cell type.

6. The method according to claim 4, wherein: further comprising identifying that the human subject has or may have damage, inflammation or cancer in the corresponding cell type.

7. The method according to claim 5, wherein: further comprising, when the amount of the cell-free DNA fragment identified as being from a pancreatic cell type is greater than a reference cut-off value, identifying that the human subject has or may have a pancreatic disease or condition.

8. The method according to claim 7, wherein: the pancreatic disease or condition is diabetes, inflammation or cancer.

9. A method for identifying that a biological sample contains DNA from a cell type, wherein: the method comprises: detecting the methylation status of each of at least four CpG sites of a target DNA fragment in the biological sample; and identifying the target DNA fragment as: (1) When no more than 40% of the CpG sites are methylated, with at least one of the CpG sites located within a human genomic sequence selected from the group consisting of SEQ ID NOs: 1793 - 1864, or when more than 50% of the CpG sites are methylated, with at least one of the CpG sites located within a human genomic sequence selected from the group consisting of SEQ ID NOs: 1893 - 1905, it is from human endometrial epithelial cells; (2) When no more than 40% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 1918 - 2022, or when more than 50% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 2043 - 2061, it is from human fallopian tube epithelial cells; (3) When no more than 40% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 2068 - 2141, or when more than 50% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 2195 - 2209, it is from human renal epithelial cells; (4) When no more than 40% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 2220 - 2298, or when more than 50% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 2346 - 2350, it is from human bladder epithelial cells; (5) When no more than 40% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 2371 - 2476, or when more than 50% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 2496 - 2500, it is from human prostate epithelial cells; (6) When no more than 40% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 2521 - 2616, or when more than 50% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 2652 - 2659, it is from human mammary gland basal epithelial cells; or (7) When no more than 40% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 2677 - 2748, or when more than 50% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 2803 - 2815, it is from human mammary gland luminal epithelial cells.

10. The method according to claim 9, wherein: The biological sample includes blood, plasma, serum, semen, milk, urine, saliva, or cerebrospinal fluid obtained from a human subject.

11. The method according to claim 10, wherein: the target DNA fragment is a cell-free DNA fragment.

12. The method according to claim 11, wherein: identifying the cell-free DNA fragment as being from a cell type indicates abnormal cell death of the cell type or a disease associated with the cell type.

13. The method according to claim 11, wherein: further comprising identifying that the human subject has or may have damage, inflammation, or cancer in the corresponding reproductive and urinary tract cells.

14. A method for identifying that a biological sample contains DNA from a cell type, wherein: the method comprises: detecting the methylation status of each CpG site among at least four CpG sites of a target DNA fragment in the biological sample; and identifying the target DNA fragment as: (1) from human alveolar epithelial cells when no more than 40% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 2828 - 2899, or when more than 50% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 2954 - 2960; (2) from human lung bronchial epithelial cells when no more than 40% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 2979 - 3087, or when more than 50% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 3105 - 3109; (3) from human heart cardiomyocytes when no more than 40% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 3130 - 3223, or when more than 50% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 3255 - 3266; (4) from human heart fibroblasts when no more than 40% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 3280 - 3394, or when more than 50% of the CpG sites are methylated and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NO: 3408 - 3414; or (5) When no more than 40% of the CpG sites are methylated, with at least one of the CpG sites being within a human genomic sequence selected from the group consisting of SEQ ID NO: 3433 - 3547, or when more than 50% of the CpG sites are methylated, with at least one of the CpG sites being within a human genomic sequence selected from the group consisting of SEQ ID NO: 3560 - 3579, it is from human vascular endothelial cells.

15. The method according to claim 14, wherein: the biological sample includes blood, plasma, serum, semen, milk, urine, saliva, or cerebrospinal fluid obtained from a human subject.

16. The method according to claim 15, wherein: the target DNA fragment is a cell-free DNA fragment.

17. The method according to claim 16, wherein: identifying the cell-free DNA fragment as being from a cell type indicates abnormal cell death of the cell type, or a corresponding cardiovascular - pulmonary cell-related disease.

18. A method for identifying that a biological sample contains DNA from a cell type, wherein: the method includes: detecting the methylation status of each of at least four CpG sites of a target DNA fragment in the biological sample; and identifying the target DNA fragment as: (1) When no more than 40% of the CpG sites are methylated, with at least one of the CpG sites being within a human genomic sequence selected from the group consisting of SEQ ID NO: 3585 - 3701, or when more than 50% of the CpG sites are methylated, with at least one of the CpG sites being within a human genomic sequence selected from the group consisting of SEQ ID NO: 3713 - 3733, it is from human B cells; (2) When no more than 40% of the CpG sites are methylated, with at least one of the CpG sites being within a human genomic sequence selected from the group consisting of SEQ ID NO: 3738 - 3849, or when more than 50% of the CpG sites are methylated, with at least one of the CpG sites being within a human genomic sequence selected from the group consisting of SEQ ID NO: 3863 - 3884, it is from human granulocytes; (3) When no more than 40% of the CpG sites are methylated, with at least one of the CpG sites being within a human genomic sequence selected from the group consisting of SEQ ID NO: 3887 - 3997, or when more than 50% of the CpG sites are methylated, with at least one of the CpG sites being within a human genomic sequence selected from the group consisting of SEQ ID NO: 4013 - 4036, it is from human monocytes or macrophages; (4) When no more than 40% of the CpG sites are methylated, and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 4038 - 4146, or when more than 50% of the CpG sites are methylated, and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 4163 - 4184, it is from human natural killer (NK) cells; (5) When no more than 40% of the CpG sites are methylated, and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 4188 - 4274, or when more than 50% of the CpG sites are methylated, and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 4313 - 4322, it is from human T cells; or (6) When no more than 40% of the CpG sites are methylated, and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 4338 - 4449, or when more than 50% of the CpG sites are methylated, and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 4465 - 4470, it is from human erythroid progenitor cells.

19. The method according to claim 18, wherein: the biological sample comprises blood, plasma, serum, semen, milk, urine, saliva or cerebrospinal fluid obtained from a human subject.

20. The method according to claim 19, wherein: the target DNA fragment is a cell-free DNA fragment.

21. The method according to claim 20, wherein: identifying the cell-free DNA fragment as being from a blood cell type indicates abnormal cell death of the cell type, or a disease associated with the blood cell type.

22. The method according to claim 20, wherein: further comprising identifying the human subject as having or likely having an autoimmune disease, inflammation, infection or cancer.

23. A method for identifying that a biological sample contains DNA from a cell type, wherein: the method comprises: detecting the methylation status of each of at least four CpG sites of a target DNA fragment in the biological sample; and identifying the target DNA fragment as: (1) When no more than 40% of the CpG sites are methylated, and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 4471 - 4573, or when more than 50% of the CpG sites are methylated, and at least one of the CpG sites is within a human genomic sequence selected from the group consisting of SEQ ID NOs: 4596 - 4598, it is from human epidermal keratinocytes; (2) When no more than 40% of the CpG sites are methylated, with at least one of the CpG sites located within a human genomic sequence selected from the group consisting of SEQ ID NO: 4619 - 4719, or when more than 50% of the CpG sites are methylated, with at least one of the CpG sites located within a human genomic sequence selected from the group consisting of SEQ ID NO: 4742 - 4747, it is from human dermal fibroblasts; (3) When no more than 40% of the CpG sites are methylated, with at least one of the CpG sites located within a human genomic sequence selected from the group consisting of SEQ ID NO: 4767 - 4869, or when more than 50% of the CpG sites are methylated, with at least one of the CpG sites located within a human genomic sequence selected from the group consisting of SEQ ID NO: 4892 - 4897, it is from human osteoblasts; (4) When no more than 40% of the CpG sites are methylated, with at least one of the CpG sites located within a human genomic sequence selected from the group consisting of SEQ ID NO: 4917 - 5016, or when more than 50% of the CpG sites are methylated, with at least one of the CpG sites located within a human genomic sequence selected from the group consisting of SEQ ID NO: 5041 - 5043, it is from human skeletal muscle cells; or (5) When no more than 40% of the CpG sites are methylated, with at least one of the CpG sites located within a human genomic sequence selected from the group consisting of SEQ ID NO: 5065 - 5178, or when more than 50% of the CpG sites are methylated, with at least one of the CpG sites located within a human genomic sequence selected from the group consisting of SEQ ID NO: 5192 - 5204, it is from human smooth muscle cells.

24. The method according to claim 23, wherein: the biological sample includes blood, plasma, serum, semen, milk, urine, saliva, or cerebrospinal fluid obtained from a human subject.

25. The method according to claim 24, wherein: the target DNA fragment is a cell-free DNA fragment.

26. The method according to claim 25, wherein: identifying the cell-free DNA fragment as being from a cell type indicates abnormal cell death of the cell type or a disease associated with the cell type.

27. The method according to claim 26, wherein: further includes identifying that the human subject has or may have inflammation or cancer in the corresponding skin - bone - muscle cells.

28. A method for identifying that a biological sample contains DNA from a cell type, wherein: the method includes: detecting the methylation status of each of at least four CpG sites of a target DNA fragment in the biological sample; and identifying the target DNA fragment as: (1) When no more than 40% of the CpG sites are methylated, with at least one of the CpG sites being within a human genomic sequence selected from the group consisting of SEQ ID NO: 5217 - 5284, or when more than 50% of the CpG sites are methylated, with at least one of the CpG sites being within a human genomic sequence selected from the group consisting of SEQ ID NO: 5344 - 5358, it is from human thyroid epithelial cells; (2) When no more than 40% of the CpG sites are methylated, with at least one of the CpG sites being within a human genomic sequence selected from the group consisting of SEQ ID NO: 5369 - 5445, or when more than 50% of the CpG sites are methylated, with at least one of the CpG sites being within a human genomic sequence selected from the group consisting of SEQ ID NO: 5454 - 5463, it is from human adipocytes; (3) When no more than 40% of the CpG sites are methylated, with at least one of the CpG sites being within a human genomic sequence selected from the group consisting of SEQ ID NO: 5471 - 5556, or when more than 50% of the CpG sites are methylated, with at least one of the CpG sites being within a human genomic sequence selected from the group consisting of SEQ ID NO: 5595 - 5613, it is from human neurons; or (4) When no more than 40% of the CpG sites are methylated, with at least one of the CpG sites being within a human genomic sequence selected from the group consisting of SEQ ID NO: 5620 - 5721, or when more than 50% of the CpG sites are methylated, with at least one of the CpG sites being within a human genomic sequence selected from the group consisting of SEQ ID NO: 5772 - 5782, it is from human oligodendrocytes.

29. The method according to claim 28, wherein: The biological sample includes blood, plasma, serum, semen, milk, urine, saliva, or cerebrospinal fluid obtained from a human subject.

30. The method according to claim 29, wherein: The target DNA fragment is a cell-free DNA fragment.

31. The method according to claim 30, wherein: Identifying the cell-free DNA fragment as being from a cell type indicates abnormal cell death of the cell type, or a disease associated with the cell type.

32. The method according to claim 30, wherein: It further includes, when the biological sample contains a target DNA fragment identified as being from oligodendrocytes, identifying that the human subject has or may have multiple sclerosis (MS).

33. The method according to claim 30, wherein: It further includes, when the biological sample contains a target DNA fragment identified as being from neurons, identifying that the human subject has or may have a neurodegenerative disorder.

34. A method for identifying that a biological sample contains DNA from lung cells, wherein: The method includes detecting the methylation status of each of at least four CpG sites of a target DNA fragment in the biological sample; and identifying that the target DNA fragment is from a human alveolar cell or a human lung bronchial cell if the methylation status corresponds to that of a reference human alveolar cell or a human lung bronchial cell, wherein according to the human genome assembly version hg19, the target DNA fragment is less than 1 kb away from a genomic locus selected from the group consisting of the following human chromosomes: chromosome 14: 55765534, chromosome 3: 181441571, chromosome 1: 41486102, chromosome 2: 236672684, chromosome 17: 79952367, chromosome 16: 678127, chromosome 7: 2473529, chromosome 16: 1652552, chromosome 14: 91691190, chromosome 16: 667157, chromosome 11: 66116455, chromosome 4: 57522145, chromosome 16: 84271391, chromosome 1: 1986275, chromosome 7: 4802132, chromosome 2: 239970075, chromosome 1: 164761834.

35. The method according to claim 34, wherein: (a) if no more than 40% of the CpG sites are methylated and the target DNA fragment is less than 1 kb away from chromosome 2: 236672684, chromosome 17: 79952367, chromosome 16: 678127, chromosome 7: 2473529, chromosome 16: 1652552, chromosome 14: 91691190, chromosome 16: 667157, chromosome 11: 66116455, chromosome 16: 84271391, or chromosome 1: 1986275, or if at least 60% of the CpG sites are methylated and the target DNA fragment is less than 1 kb away from chromosome 4: 57522145, then the target DNA fragment is identified as being from a human alveolar cell; (b) if no more than 40% of the CpG sites are methylated and the target DNA fragment is less than 1 kb away from chromosome 7: 4802132, chromosome 2: 239970075, or chromosome 1: 164761834, then the target DNA fragment is identified as being from a human lung bronchial cell; or (c) if no more than 40% of the CpG sites are methylated and the target DNA fragment is less than 1 kb away from chromosome 14: 55765534 or chromosome 1: 41486102, or if at least 60% of the CpG sites are methylated and the target DNA fragment is less than 1 kb away from chromosome 3: 181441571, then the target DNA fragment is identified as being from a human alveolar cell or a human lung bronchial cell.

36. The method according to any one of claims 1 to 35, wherein: The length of the target DNA fragment is 50 bp to 200 bp.

37. The method according to any one of claims 1 to 36, wherein: the methylation state is the conversion of cytosine to 5-methylcytosine (5-mC) or 5-hydroxymethylcytosine (5-hmC).

38. The method according to claim 37, wherein: detecting the methylation state includes treating the DNA fragment with bisulfite or an enzyme, or digesting the DNA fragment with a restriction endonuclease sensitive to DNA methylation.

39. The method according to claim 38, wherein: the enzyme treatment includes treatment with APOBEC-Seq.

40. The method according to claim 38, wherein: detecting the methylation state further includes determining the sequence of the DNA fragment.

41. The method according to claim 40, wherein: the sequence is determined by deep sequencing.

42. The method according to claim 38, wherein: detecting the methylation state further includes detecting the digested fragments.

43. The method according to any one of claims 1 to 42, wherein: further includes detecting genetic variations in the target DNA fragment, so as to determine that the cell releasing the target DNA fragment contains the genetic variations.

44. The method according to any one of claims 5 to 8, 12 to 13, 17, 21 to 22, 26 to 27, and 32 to 33, wherein: further includes administering to the patient an agent that can be used to treat the identified disease or condition.

45. A method for determining the cell type of cancer cells, wherein: the method includes identifying the cell type of the cancer cells by the method according to any one of claims 1 to 44.

46. The method according to claim 45, wherein: further includes detecting genetic variations in the genomic DNA of the cancer cells.

47. The method according to claim 45 or 46, wherein: the cancer cells are obtained from a biological sample selected from the group consisting of blood, plasma, serum, semen, milk, urine, saliva, or cerebrospinal fluid.

48. The method according to claim 47, wherein: further includes localizing the tissue origin of the cancer cells based on the cell type.

49. The method according to any one of claims 45 to 48, wherein: further includes treating cancer in a subject from whom the cancer cells are obtained.

Images