Compositions and methods for identifying cell types

Abstract

The present disclosure generally relates to compositions and methods for determining cell types based on the methylation profile of the associated DNA. For cell-free DNA, such determination can be used to identify diseases or conditions associated with the cell type. For tumor cells, such determination is useful for identifying their primary source.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit under 35 USC 119(e) of U.S. Provisional Patent Application No. 63 / 295,319, filed December 30, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (334655 WO.xml; size: 13,761,379 bytes; created: December 27, 2022) are incorporated herein by reference in their entirety. [Background technology]

[0003] Identifying the origin of cells or cell-free DNA is important. For example, tumor cells may migrate to other tissues, making their origin difficult to identify. Cancer of unknown primary origin (CUP) is cancer that has been determined to be at a metastatic stage at the time of diagnosis, but for which the primary tumor cannot be identified. CUP is found in approximately 3-5% of all people diagnosed with invasive cancer, and in most (80-85%) of these cases, the prognosis is poor.

[0004] Small fragments of nucleic acids, such as DNA, circulate freely in the peripheral blood of healthy and diseased individuals. These cell-free nucleic acids, such as DNA (cfDNA), may originate from dying or damaged cells and thus reflect ongoing cell death or damage occurring in the body. In recent years, this understanding has led to the emergence of diagnostic tools that have impacted multiple areas of medicine. For example, next-generation sequencing of fetal DNA circulating in maternal blood has enabled noninvasive prenatal testing for fetal chromosomal abnormalities. Detection of donor-derived DNA in the circulation of organ transplant recipients can be used for early identification of transplant rejection, and assessment of circulating mutant DNA can be used for genotyping and cancer monitoring.

[0005] While such techniques are powerful for identifying genetic abnormalities in circulating DNA or translocated cells, they are not informative when the DNA does not carry mutations. A key limitation of sequencing is that it does not reveal the tissue origin of the DNA, preventing the identification of tissue-specific cancer or cell death. The latter is important in many situations, such as neurodegenerative, inflammatory, or ischemic diseases that do not involve DNA mutations. Even in oncology, for example, in the context of CUP and early cancer diagnosis, it is often important to determine the tissue origin of a tumor in addition to determining its mutation profile.

[0006] Identification of the tissue origin of DNA may also provide insight into concomitant tissue damage (eg, drug toxicity in genetically normal tissue), an important factor in drug development and monitoring treatment response. Summary of the Invention

[0007] The present disclosure provides compositions and methods for determining cell types based on the methylation status of DNA fragments. Also provided are compositions and methods for identifying diseases and conditions in a subject, e.g., a human subject, through cell-free DNA released by cells affected by such diseases or conditions. In oncology or other disease states, this technology can be used to identify the primary origin of tumor cells.

[0008] In one embodiment, the present disclosure provides a method for identifying that a biological sample contains 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 epithelium, kidney epithelium, bladder epithelium, prostate epithelium, breast basal epithelium, breast luminal epithelium, alveolar epithelium, lung bronchial epithelium, cardiac cardiomyocytes, cardiac fibroblasts, vascular endothelial cells, blood B cells, blood granulocytes, blood monocytes + macrophages, blood NK cells, blood T cells, erythroid precursor cells, epidermal keratinocytes, skin fibroblasts, osteoblasts, skeletal muscle cells, smooth muscle cells, thyroid epithelium, adipocytes, neural CNS cells, and oligodendrocytes.

[0009] In some embodiments, the methods involve detecting the methylation state of at least four, or each of at least five, six, seven, or eight CpG sites of a target DNA fragment in a biological sample, and identifying the target DNA fragment as being from a human cell type if the methylation state of the target DNA fragment corresponds to the methylation state of the DNA fragment defined in Table A for that cell type.

[0010] As used herein, in some embodiments, methylation status refers to the percentage of CpG sites that are methylated within a target DNA fragment (e.g., 25%). In some embodiments, methylation status refers to whether a target DNA fragment is hypermethylated (M, at least 60% CpG methylation) or hypomethylated (U, 40% or less CpG methylation) compared to the same fragment in other cell types.

[0011] The target DNA fragments, in some embodiments, have the DNA sequences shown in the attached Table B and Sequence Listing. However, as shown in the experimental examples, the methylation patterns are uniform across the contiguous region. Therefore, the sequences or their genomic locations are representative of nearby genomic regions.

[0012] In some embodiments, the target DNA fragment comprises at least a CpG site within a sequence included in the sequence listing. In some embodiments, the target DNA fragment comprises at least two CpG sites within a sequence included in the sequence listing. In some embodiments, the target DNA fragment comprises at least three or four CpG sites within a sequence included in the sequence listing.

[0013] In some embodiments, the target DNA fragment is within 1000 bp of either the 5' or 3' end of a sequence included in the sequence listing. In some embodiments, the target DNA fragment is within 900, 800, 700, 600, 500, 400, 300, 250, 200, or 150 bp of either the 5' or 3' end of a sequence included in the sequence listing.

[0014] In some embodiments, the target DNA fragments are obtained from a biological sample selected from the group consisting of blood, plasma, serum, semen, milk, urine, saliva, and cerebrospinal fluid.

[0015] In some embodiments, the target DNA fragments are cell-free DNA fragments. In some embodiments, identifying the cell-free DNA fragments as being from a cell type comprises detecting aberrant cell death of the cell type or a disease associated with the cell type. In some embodiments, the method further involves identifying the human subject as having or likely to have damage, inflammation, or cancer in the corresponding cell type.

[0016] In some embodiments, the disease or condition is physical injury, inflammation, infection, cancer, diabetes, an autoimmune disease, multiple sclerosis (MS), or a neurodegenerative disorder.

[0017] In some embodiments, the target DNA fragment has a length of 20 to 500 bp, including, but not limited to, 30 to 400 bp, 40 to 300 bp, 50 to 250 bp, 50 to 200 bp, or 50 to 150 bp.

[0018] In some embodiments, the methylation status is conversion of cytosine to 5-methylcytosine (5-mC) or 5-hydroxymethylcytosine (5-hmC). In some embodiments, detecting the methylation status comprises bisulfite or enzymatic treatment of the DNA fragment, or digestion of the DNA fragment with a restriction enzyme sensitive to DNA methylation. In some embodiments, the enzymatic treatment comprises treatment with APOBEC-Seq. In some embodiments, detecting the methylation status further comprises determining the sequence of the DNA fragment. In some embodiments, the sequence is determined by deep sequencing.

[0019] In some embodiments, the method further comprises detecting a genetic mutation in the target DNA fragment, thereby determining that the cell into which the target DNA fragment is released contains the genetic mutation. In some embodiments, the method further comprises administering to the patient an agent useful for treating the identified disease or condition. [Brief explanation of the drawings]

[0020] [Figure 1] An adult human methylation atlas is shown. 207 healthy samples were obtained from adults, isolated, and deep sequenced (WGBS, average depth >30x) to form a comprehensive human cell type-specific methylation atlas. [Figure 2] Figure 1 shows the segmentation of the human genome into 7,264,350 contiguous uniform blocks. The histograms show the number of segmented blocks as a function of their base length (left) or the number of CpGs they contain (right). In addition to the 2,746,623 blocks of CpGs between 3 and 30 in length (plotted at the top), there were also 3,271,607 blocks of one CpG, 1,185,719 blocks of two CpGs, and 60,401 blocks of more than 30 CpGs. [Figure 3]We show that biological replicates of the same cell type from different individuals exhibit a surprisingly low percentage of differentially methylated blocks. We focused on 37 cell subtypes (e.g., endothelial cells from specific tissues) with n ≥ 3 replicates and measured the average percentage of methylated blocks (≥ 3 CpGs) whose methylation differed by 50% (absolute delta-beta) across replicates (shown as the y-axis). Nearly all cell subtypes (36 / 37) differed by 0.5% or less of the blocks, suggesting a very high degree of conservation between replicates. The red dotted line indicates the average number of differential blocks between two random samples of different cell types (4.9%). [Figure 4] We show that unsupervised agglomerative clustering reflects the human developmental lineage of healthy cell types. [Figure 5] The average methylation in the top differentially methylated blocks is shown. The average methylation value in the 1% of the most variable blocks with four or more CpGs (21,077 blocks) is shown. For each block, the average methylation in each sample was calculated and classified as unmethylated (<50%) or methylated (>50%). Box plots show the 25th to 75th percentiles of the average methylation levels in unmethylated blocks / samples (blue), methylated ones (yellow), or the difference between methylated and unmethylated samples within the same block (green). [Figure 6-1]A human methylation atlas of 207 samples across 39 cell types is shown. (A) 953 genomic regions that are unmethylated in a cell-type-specific manner. Each cell in the plot represents the average methylation of one genomic region (column) in each of the 39 cell types (rows). Up to 25 regions per cell type are shown, with an average length of 251 bp (9 CpGs) per region. (B) The top 25 cardiomyocyte regions. For each region, the average methylation of each CpG site (column) across all 207 samples is plotted in the atlas and categorized into the 39 cell types as described above. (C) Genetic loci that are specifically unmethylated in cardiomyocytes. This marker (highlighted in light blue) is 120 bp long (6 CpGs) and located in the first intron of the cardiac-specific gene MYL4 (2518 TPM expression in the atrial appendage, GTEx inset). Genome snapshots show average methylation (purple tracks) across 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 mapping to chr17:45289451-45289570 (hg19) with at least three covered CpGs are shown. Yellow / blue dots indicate methylated / unmethylated CpG sites. [Figure 6-2]A human methylation atlas of 207 samples across 39 cell types is shown. (A) 953 genomic regions that are unmethylated in a cell-type-specific manner. Each cell in the plot represents the average methylation of one genomic region (column) in each of the 39 cell types (rows). Up to 25 regions per cell type are shown, with an average length of 251 bp (9 CpGs) per region. (B) The top 25 cardiomyocyte regions. For each region, the average methylation of each CpG site (column) across all 207 samples is plotted in the atlas and categorized into the 39 cell types as described above. (C) Genetic loci that are specifically unmethylated in cardiomyocytes. This marker (highlighted in light blue) is 120 bp long (6 CpGs) and located in the first intron of the cardiac-specific gene MYL4 (2518 TPM expression in the atrial appendage, GTEx inset). Genome snapshots show average methylation (purple tracks) across 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 mapping to chr17:45289451-45289570 (hg19) with at least three covered CpGs are shown. Yellow / blue dots indicate methylated / unmethylated CpG sites. [Figure 7] Cell-type specific markers enriched for regulatory motifs. Using HOMER motif analysis, we show the top transcription factor binding site motifs enriched among the top 250 differentially unmethylated regions per cell type. Motifs similar to previous (more significant) hits are skipped. [Figure 8-1]Cell-type-specific hypermethylated regions are shown to be enriched for CpG islands, Polycomb targets, and CTCF and REST / NSRF. (A) 37.9% (1,185 of 3,125, p<1E-100) of the top cell-type-specific hypermethylated markers overlap with CpG islands. For comparison, 1.7% (198 / 11,371, p<2E-29) of the cell-type-specific hypomethylated regions overlap with CpG islands, which comprise less than 0.9% of the genome (black line). (B) These regions are typically enriched for H3K27me3 in other cell types. Average H3K27me3 signal in monocytes and macrophages near all cell-type-specific hypermethylated regions (top, blue) or near monocyte / macrophage-specific hypermethylated regions (green) is shown. (C) Similar plots for Polycomb annotation in monocytes and macrophages (chromHMM) for all or monocyte / macrophage-specific markers. (D) Motif analysis of cell-type-specific hypermethylated regions (top 100 per cell type) identifies known CTCF and REST / NSRF motifs. (E) Analysis of ChIP-seq data for one such site (chr1:209364093-209364250, highlighted in blue, hg19) that is specifically methylated in the small intestine and colon epithelium (Box 1) and unmethylated elsewhere. As shown below, this site is bound in multiple cell types and tissues, but is largely unbound in vivo in the gastric and colonic epithelium (Box 2). (F) REST / NSRF motifs are present within 15 (15%) of the top 100 cell type-specific hypermethylated regions in the endocrine pancreas (alpha, beta, and delta cells), within 5 of the top 100 pancreatic delta cells, and within 2 of the top 100 pancreatic beta cells, in accordance with REST target expression in the endocrine pancreas, compared to ~0.1% in background sequences. [Figure 8-2]Cell-type-specific hypermethylated regions are shown to be enriched for CpG islands, Polycomb targets, and CTCF and REST / NSRF. (A) 37.9% (1,185 of 3,125, p<1E-100) of the top cell-type-specific hypermethylated markers overlap with CpG islands. For comparison, 1.7% (198 / 11,371, p<2E-29) of the cell-type-specific hypomethylated regions overlap with CpG islands, which comprise less than 0.9% of the genome (black line). (B) These regions are typically enriched for H3K27me3 in other cell types. Average H3K27me3 signal in monocytes and macrophages near all cell-type-specific hypermethylated regions (top, blue) or near monocyte / macrophage-specific hypermethylated regions (green) is shown. (C) Similar plots for Polycomb annotation in monocytes and macrophages (chromHMM) for all or monocyte / macrophage-specific markers. (D) Motif analysis of cell-type-specific hypermethylated regions (top 100 per cell type) identifies known CTCF and REST / NSRF motifs. (E) Analysis of ChIP-seq data for one such site (chr1:209364093-209364250, highlighted in blue, hg19) that is specifically methylated in the small intestine and colon epithelium (Box 1) and unmethylated elsewhere. As shown below, this site is bound in multiple cell types and tissues, but is largely unbound in vivo in the gastric and colonic epithelium (Box 2). (F) REST / NSRF motifs are present within 15 (15%) of the top 100 cell type-specific hypermethylated regions in the endocrine pancreas (alpha, beta, and delta cells), within 5 of the top 100 pancreatic delta cells, and within 2 of the top 100 pancreatic beta cells, in accordance with REST target expression in the endocrine pancreas, compared to ~0.1% in background sequences. [Figure 9-1]Results of lung epithelial methylome analysis are shown. A. Comparative tissue methylome analysis reveals multiple methylation blocks that are uniquely unmethylated in alveoli (1,663 blocks), bronchial epithelial cells (673 blocks), or both (139 blocks) and methylated in all other tissues. An additional 11 markers specifically methylated in lung are not shown. Each marker encompasses three or more CpGs and exhibits a mean methylation delta of 0.4 or greater between the 25th percentile of the target cell type and the 97.5th percentile of other tissues. B. Characterization of one alveolar-specific methylation marker located at chr16:667119-667272 (hg19) in the Rab40C gene. This region is unmethylated exclusively in alveolar epithelium and is enriched for the chromatin markers H3K27ac, H3K4me1, and H3K4me3. C. Lung-specific methylation markers are enriched for enhancer regions. For each of the three marker sets, the number of markers with enhancer-associated chromatin states in lung is shown, showing enrichment of fold changes between 2.5 and 10. D. GREAT annotation identifies enriched gene sets among genes closest to lung-unique methylation markers. Five of the most significant (BinomFDRQ) gene sets for the methylation markers in each lung cell type are shown. [Figure 9-2]Results of lung epithelial methylome analysis are shown. A. Comparative tissue methylome analysis reveals multiple methylation blocks that are uniquely unmethylated in alveoli (1,663 blocks), bronchial epithelial cells (673 blocks), or both (139 blocks) and methylated in all other tissues. An additional 11 markers specifically methylated in lung are not shown. Each marker encompasses three or more CpGs and exhibits a mean methylation delta of 0.4 or greater between the 25th percentile of the target cell type and the 97.5th percentile of other tissues. B. Characterization of one alveolar-specific methylation marker located at chr16:667119-667272 (hg19) in the Rab40C gene. This region is unmethylated exclusively in alveolar epithelium and is enriched for the chromatin markers H3K27ac, H3K4me1, and H3K4me3. C. Lung-specific methylation markers are enriched for enhancer regions. For each of the three marker sets, the number of markers with enhancer-associated chromatin states in lung is shown, showing enrichment of fold changes between 2.5 and 10. D. GREAT annotation identifies enriched gene sets among genes closest to lung-unique methylation markers. Five of the most significant (BinomFDRQ) gene sets for the methylation markers in each lung cell type are shown. [Figure 10-1]Performance of selected lung-specific markers is shown. A. Assay specificity. Methylation status of lung epithelial markers (alveoli in green, bronchi in orange, and general lung in pink) in DNA from multiple tissues. The percentage of molecules with most CpG sites methylated or unmethylated is shown. B. Assay specificity in lung cancer. Methylation status of lung epithelial markers in DNA from multiple lung cancers. The percentage of molecules with most CpG sites methylated or unmethylated according to the marker is shown. 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. Assay sensitivity and precision in vitro. DNA from healthy human alveolar (left) or bronchial (right) epithelium was mixed with blood DNA as indicated, and the fraction of methylated or unmethylated molecules in the lung markers was determined. Assay robustness. Duplicate cfDNA samples extracted from the same donor were analyzed for lung markers. The number of genome equivalents per mL of plasma present in each duplicate is shown. [Figure 10-2] Performance of selected lung-specific markers is shown. A. Assay specificity. Methylation status of lung epithelial markers (alveoli in green, bronchi in orange, and general lung in pink) in DNA from multiple tissues. The percentage of molecules with most CpG sites methylated or unmethylated is shown. B. Assay specificity in lung cancer. Methylation status of lung epithelial markers in DNA from multiple lung cancers. The percentage of molecules with most CpG sites methylated or unmethylated according to the marker is shown. 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. Assay sensitivity and precision in vitro. DNA from healthy human alveolar (left) or bronchial (right) epithelium was mixed with blood DNA as indicated, and the fraction of methylated or unmethylated molecules in the lung markers was determined. Assay robustness. Duplicate cfDNA samples extracted from the same donor were analyzed for lung markers. The number of genome equivalents per mL of plasma present in each duplicate is shown. [Figure 11] Figure 1 shows the results of a study of lung-derived cfDNA in healthy individuals. A. Lung cfDNA concentrations in the plasma of 30 healthy donors. Concentrations were measured by multiplying the fraction of lung cfDNA by the concentration of total cfDNA. B. Lung cfDNA fraction in the plasma of 30 healthy donors and in lung lavage fluid of 6 donors. [Figure 12-1] Identification of lung-derived cfDNA in lung cancer patients. A. Lung cfDNA in the plasma of 26 patients with advanced lung cancer. Concentrations were measured by multiplying the fraction of lung cfDNA by the concentration of total cfDNA. The dashed lines in this panel and in C indicate the mean + 2 standard errors of the mean for healthy controls. B. Lung cfDNA in the plasma of patients with lung cancer. Top: P values ​​were determined by a two-sided Mann-Whitney test. Bottom: ROC curve for all advanced lung cancer patients versus healthy samples. C. Lung cfDNA in the plasma of 51 donors who underwent bronchoscopy. Concentrations were measured by multiplying the fraction of lung cfDNA by the concentration of total cfDNA. P values ​​were determined by a two-sided Mann-Whitney test. Left: Each color represents the cumulative value of the marker for the indicated cell type. Right: Each dot represents the cumulative value of all measured lung markers. D. Concentration of lung cfDNA in plasma of donors undergoing bronchoscopy versus healthy patients (left), and ROC curve for distinguishing patients with lung pathology from healthy controls. [Figure 12-2]Identification of lung-derived cfDNA in lung cancer patients. A. Lung cfDNA in the plasma of 26 patients with advanced lung cancer. Concentrations were measured by multiplying the fraction of lung cfDNA by the concentration of total cfDNA. The dashed lines in this panel and in C indicate the mean + 2 standard errors of the mean for healthy controls. B. Lung cfDNA in the plasma of patients with lung cancer. Top: P values ​​were determined by a two-sided Mann-Whitney test. Bottom: ROC curve for all advanced lung cancer patients versus healthy samples. C. Lung cfDNA in the plasma of 51 donors who underwent bronchoscopy. Concentrations were measured by multiplying the fraction of lung cfDNA by the concentration of total cfDNA. P values ​​were determined by a two-sided Mann-Whitney test. Left: Each color represents the cumulative value of the marker for the indicated cell type. Right: Each dot represents the cumulative value of all measured lung markers. D. Concentration of lung cfDNA in plasma of donors undergoing bronchoscopy versus healthy patients (left), and ROC curve for distinguishing patients with lung pathology from healthy controls. [Figure 13] Figure 1 shows the effect of the number of lung markers on assay sensitivity. A. ROC curve using the indicated combinations of lung methylation markers to identify patients with any lung pathology vs. healthy controls. B. Sensitivity of the indicated combinations of lung markers at 70% specificity. Patients with lung pathology vs. healthy controls. [Figure 14] Results of a study of lung-specific cfDNA in patients with COPD. A. Lung cfDNA concentrations in the plasma of 77 patients with COPD. Concentrations were measured by multiplying the fraction of lung cfDNA by the concentration of total cfDNA. The dashed line indicates the mean + 2 standard errors of the mean for healthy controls. B. Lung cfDNA in the plasma of patients with lung cancer, exacerbations, and stable COPD, as well as healthy controls. C. Lung cfDNA in the plasma of COPD patients who were still alive 14 months after sampling versus patients who died during this period. [Figure 15] FIG. 1 is a schematic diagram illustrating computing components that may be used to implement various features of the embodiments described in this disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0021] The following description describes exemplary embodiments of the present technology, however, it should be recognized that such description is not intended as a limitation on the scope of the present disclosure, but is instead provided as a description of exemplary embodiments.

[0022] definition Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this description belongs. As used herein, the following terms have the meanings ascribed to them below.

[0023] 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 to form 5-methylcytosine. The term also includes the process by which a hydroxymethyl group is attached to a DNA molecule, e.g., by oxidation of a methyl group on the pyrimidine ring of a cytosine base (specifically, "hydroxymethylation"). Methylation, including hydroxymethylation, generally occurs at dinucleotides of cytosine and guanine, referred to herein as "CpG dinucleotides" or "CpG sites." The principles described herein are also applicable to detecting methylation in non-CpG contexts, including non-cytosine methylation. In such embodiments, the wet-laboratory assay used to detect methylation may differ from any described herein. Furthermore, a methylation state vector may generally include elements that are vectors of methylated or unmethylated sites (even if these sites are not specifically CpG sites).

[0024] As used herein, the term "methylation site" refers to a region of a DNA molecule where a methyl group can be attached to the DNA molecule. Although "CpG" sites are the most common methylation sites, methylation sites are not limited to CpG sites. For example, DNA methylation can occur at cytosine in CHG and CHH (where H is adenine, cytosine, or thymine).

[0025] As used herein, the term "CpG site" refers to a region of a DNA molecule in which a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases along its 5' to 3' direction. "CpG" is an abbreviation for 5'-C-phosphate-G-3', in which the cytosine and guanine are separated by only one phosphate group. The cytosine in a CpG dinucleotide can be methylated to form 5-methylcytosine.

[0026] As used herein, the terms "hypomethylated" or "hypermethylated" refer to the methylation state of a DNA molecule containing multiple CpG sites (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more, etc.), where a higher percentage of CpG sites (e.g., 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more) are methylated compared to corresponding DNA molecules from one or more reference samples. The ranges provided herein may be 0% to 50% or greater, 40% or greater, 50% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, or 95% or greater, or 97.5% or greater, 98% or greater, 99% or greater, or 99.9% or greater, or any other numerical percentage within the range of 0% to 50% or the range of 50% to 100%, where each range provided herein indicates that the range endpoints (e.g., 50% and 100%) are unmethylated or methylated, respectively. In the context of cancer, the reference sample may be normal tissue. Hypomethylation of DNA molecules from tumor cells refers to a decreased percentage of methylation compared to normal, e.g., healthy, non-diseased, e.g., non-cancerous, tissue, also known as "hypomethylation." A "hypomethylated" nucleic acid, e.g., cfDNA fragment, can be a fragment having a number, e.g., 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more CpG sites, in which a percentage, e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more, or 97.5% or more, 98% or more, 99% or more, 99.9% or more of the CpG sites are unmethylated. Hypermethylation of DNA molecules from tumor cells refers to an increased percentage of methylation compared to normal, e.g., healthy, non-diseased, e.g., non-cancerous tissue, and is also known as "hypermethylation."Similarly, a "hypermethylated" nucleic acid, e.g., cfDNA fragment, can be a fragment having a certain number of CpG sites, e.g., 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more, in which a certain percentage, e.g., 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more, or 97.5% or more, 98% or more, 99% or more, 99.9% or more of the CpG sites are methylated. "Hypomethylation" can also refer to a lower percentage of methylated DNA molecules in a target cell compared to other types of cells, and hypermethylation can also refer to a higher percentage of methylated DNA molecules in a target cell compared to other types of cells.

[0027] The term "cell-free nucleic acid" refers to nucleic acids, such as DNA in "cell-free DNA" and "cfDNA" fragments, that circulate within an individual's body (e.g., the bloodstream) and are derived from one or more healthy cells and / or one or more diseased, senescent, or damaged cells. In addition, cell-free nucleic acids, such as cfDNA, can be derived from other sources, such as viruses, fetuses, etc.

[0028] The terms "circulating tumor DNA" and "ctDNA" refer to DNA fragments derived from tumor cells, which may be released into an individual's bloodstream as a result of biological processes such as apoptosis or necrosis of dying cells, or may be actively released by viable tumor cells.

[0029] As used herein, the terms "aberrant methylation pattern" and "atypical methylation pattern" refer to a methylation pattern of a nucleic acid, e.g., a DNA molecule such as cfDNA, or a methylation status vector, that is found and / or expected to be found in a sample at a lower frequency than a healthy, e.g., non-cancer, sample. In various embodiments, such a methylation pattern is found and / or expected to be found in a sample at a lower frequency than a value, e.g., a threshold, for a non-cancer or healthy, e.g., non-cancer, sample. Thus, for example, as used herein, the terms "aberrantly methylated" and "atypically methylated" describe a nucleic acid, e.g., a DNA molecule such as cfDNA, or a methylation status vector, that exhibits an abnormal methylation pattern. Aspects according to the present disclosure that are differentially methylated can, in some versions, include aspects that are aberrantly methylated. Furthermore, whether an aspect is differentially methylated can be used as an indicator of the health of the subject from which the sample is derived, to determine whether the subject is healthy, e.g., non-cancer, as opposed to diseased, e.g., cancerous. In some versions, the method includes determining whether the nucleic acid, e.g., a DNA molecule or methylation status vector, is aberrantly methylated.

[0030] As used herein, the term "methylation status vector" refers to a vector containing multiple elements, each element representing, in the order of their appearance 5' to 3' in the DNA molecule, a nucleic acid containing multiple methylation sites, e.g., the methylation status of methylation sites in the DNA molecule. <M x ,M x+1 ,M x+2 >, <M x ,M x+1 ,U x+2 >,..., x ,U x+1 ,U x+2 > can be a methylation vector for a DNA molecule containing three methylation sites, where M represents a methylated methylation site and U represents an unmethylated methylation site.

[0031] ​The terms "converted DNA molecule" and "converted cfDNA molecule" refer to DNA, e.g., cfDNA molecules, obtained by treating molecules in a sample to distinguish between methylated and unmethylated nucleotides in the DNA or cfDNA molecule. For example, in one embodiment, the sample can undergo bisulfite conversion, thereby treating 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 an enzymatic conversion reaction, e.g., a reaction using cytidine deaminase (such as APOBEC). After treatment, the converted DNA molecule or cfDNA molecule contains additional uracil not present in the original cfDNA sample. Replication of the uracil-containing DNA strand by DNA polymerase results in the addition of adenine 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.

[0032] The term "converted DNA sequence" refers to the sequence of a converted DNA molecule.

[0033] As used herein, the term "tissue of origin" or "TOO" refers to the organ, group of organs, body region, and / or cell type from which nucleic acids, e.g., cfDNA, e.g., healthy or disease-related, e.g., cancer-related cfDNA, are derived. Identification of the tissue of origin and / or disease, e.g., cancer, cell type allows for identification of the most appropriate next steps in the disease treatment continuum and can further diagnose, stage, and determine treatment.

[0034] Identification of cell types based on DNA methylation status The present disclosure provides compositions and methods for determining cell types based on the methylation status of related DNA fragments. Such DNA fragments typically possess multiple adjacent CpG dinucleotides with a relatively uniform methylation status (methylated or unmethylated) within a cell type. Meanwhile, the methylation status of such CpG sites varies among other cells, thereby allowing each 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."

[0035] Previously, DNA methylation analysis has primarily used bulk tissues and measured average methylation across probed CpG sites, thus precluding the study of a small number of cell types in which DNA methylation may differ, such as tissue-resident immune cells, fibroblasts, or endothelial cells. Alternatively, analysis of cultured cells often suffers from the inherent limitations of non-physiological methylation patterns introduced in vitro.

[0036] To overcome these limitations and accurately characterize the complexity of the human cellular methylome, we isolated FACS-purified populations of 39 primary human cell types from freshly dissociated adult healthy tissues. Unlike many previous studies that used shallow sequencing or were limited to a subset of genomic regions (reduced representation bisulfite-sequencing, RRBS), this study employed deep genome-wide sequencing using paired-end reads at an average sequencing depth of 32× (±7.2×) in purified human cell populations. For each cell type, the analysis included multiple replicates obtained from different individuals. The analysis coalesced genome-wide read-specific methylation patterns into larger blocks, allowing for simultaneous readout of the methylation status of multiple CpG sites, capturing dependencies between neighboring CpG sites while reflecting the variance of methylation patterns across individual cell types.

[0037] As demonstrated in the accompanying experimental examples, surprisingly, in all of the multiple human cell types tested, a sufficient number of CpG clusters could be identified as having a statistically different methylation state between the cell type and all other cell types. Such CpG clusters, also called "methylation markers," allow the identification of each cell type based on its DNA methylation state.

[0038] 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 involves detecting the methylation status of multiple CpG sites in a DNA fragment and identifying the corresponding cell type based on the methylation status of the sites. According to various embodiments, the DNA fragment is derived from one or more cells of the determined cell type.

[0039] Methylation detection Detection of DNA methylation according to this embodiment can be carried out in a variety of ways. 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).

[0040] In some embodiments, the methylation status is detected directly, such as using mass spectrometry or methylation-sensitive restriction enzymes. Steps in DNA methylation methods can produce converted DNA molecules. In such embodiments, methylated cytosines are converted prior to further analysis. The terms "converting" and "modifying" refer to treating DNA molecules in a sample for the purpose of distinguishing between methylated and unmethylated nucleotides. For example, in one embodiment, a sample can be treated with bisulfite ions (e.g., using sodium bisulfite) to convert unmethylated cytosines ("C") to uracil ("U"). In another embodiment, conversion of unmethylated cytosines to uracil is achieved using an enzymatic conversion reaction, e.g., using cytidine deaminase, such as APOBEC-Seq (NEBiolabs, Ipswich, MA). Exemplary methods for detecting DNA methylation are further described below.

[0041] Methylation-specific PCR (MSP) can be based on the chemical reaction of sodium bisulfite with DNA, which converts unmethylated cytosines in CpG dinucleotides to uracil or UpG, followed by conventional PCR. Methylated cytosines are not converted in this process, and primers are designed to overlap the CpG sites of interest, which allows the methylation status to be determined as methylated or unmethylated.

[0042] Whole-genome bisulfite sequencing, also known as BS-Seq, is a high-throughput genome-wide analysis of DNA methylation. It can also be based on sodium bisulfite conversion of genomic DNA, which is then sequenced on a next-generation sequencing platform such as deep sequencing. The resulting sequences are then aligned to a reference genome to determine the methylation status of CpG dinucleotides based on mismatches resulting from the conversion of unmethylated cytosines to uracils.

[0043] The HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) assay compares representations generated by digestion of the genome with a restriction enzyme, e.g., HpaII or MspI, followed by ligation-mediated PCR. HpaII digests 5'-CCGG-3' sites when the cytosine in the central CG dinucleotide is unmethylated, and the HpaII representation is enriched for the hypomethylated fraction of the genome.

[0044] The Glal hydrolysis and ligation adapter dependent PCR (GLAD-PCR) assay can determine R(5mC)GY sites generated during de novo DNA methylation by DNMTЗA and DNMTЗB DNA methyltransferases. GLAD-PCR does not require bisulfite treatment of DNA. GLAD-PCR uses a site-specific methyl-directed DNA endonuclease (MD DNA endonuclease) that cleaves only methylated DNA and not unmethylated DNA.

[0045] Illumina methylation assays measure locus-specific DNA methylation using array hybridization. Bisulfite-treated DNA is hybridized to probes on BeadChips. Single-base extension with 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 human genome.

[0046] The "Enzymatic Methyl-seq" or "EM-seq" method, developed at New England Biolabs, offers an alternative to bisulfite modification. This method relies on the ability of APOBEC (e.g., APOBEC-Seq with NEB) to deaminate cytosine to uracil. Cytosine is then sequenced as thymine, and methylated cytosine is sequenced as cytosine.

[0047] DNA sample preparation DNA fragments to be subjected to methylation status detection can be prepared from cell-containing or cell-free samples. Cell-containing biological samples can be easily obtained from, but are not limited to, biopsies, cultured cells, skin tissue, cells, body fluids, etc. 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 subjected to the present method include blood, plasma, serum, semen, milk, urine, vaginal fluid, uterine or vaginal lavage fluid, multiple fluids, ascites, sweat, tears, sputum, bronchoalveolar lavage fluid, feces, saliva, and cerebrospinal fluid.

[0048] In some embodiments, cell-free DNA samples can also be used. Cell-free DNA circulates within an individual's body and can be derived from healthy cells or diseased, aged, or damaged cells. In the case of pregnancy, cell-free DNA can be derived from the fetus. In some embodiments, cell-free DNA is obtained from biological samples including blood, plasma, serum, semen, milk, urine, saliva, or cerebrospinal fluid, or any other bodily fluid or tissue.

[0049] DNA fragments can be isolated from biological samples using methods known in the art. In some embodiments, the DNA fragments are substantially free of proteins, lipids, and other common materials from tissue or fluid samples. In some embodiments, the DNA fragments have a length suitable for methylation analysis.

[0050] In some embodiments, the DNA fragments have an average length of at least 18, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, or 350 bp. In some embodiments, the DNA fragments have an average length of less than 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, or 350 bp. In some embodiments, the DNA fragments have an average length of, but not limited to, 40 to 300, 400 to 250, 40 to 200, 50 to 300, 50 to 250, 50 to 200, 50 to 150, 100 to 300, 100 to 250, 100 to 200, or 150 to 300 bp.

[0051] In some embodiments, DNA fragments from a 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 concentrating DNA fragments of a desired length, while discarding DNA fragments that are too short or too long, such as by liquid chromatography.

[0052] In some embodiments, degradation of DNA fragments is not required even if their average length is longer than desired. Alternatively, detection of DNA methylation can be limited to desired fragments / sequences using suitable primer design (e.g., methylation-specific PCR) or targeted mapping of detected methylation states within the desired fragments / sequences.

[0053] Methylation detection can be performed on the prepared DNA fragments. In some embodiments, it is desirable to detect the methylation status of adjacent CpG sites that collectively form a CpG cluster. As used herein, the term "adjacent" refers to two or more CpG sites, all of which are located within a region on the DNA fragment. In some embodiments, the region has a length of less than 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, or 500 bp. In some embodiments, a CpG site is considered to be adjacent to another CpG site if the distance between them is less than 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, or 500 bp.

[0054] 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, 11, 12, 13, 14, or 15 CpG sites is detected. Each such site may be completely or partially non-adjacent to other sites, for example, a site may be adjacent to another site on one side and not adjacent to the other site on the opposite side, or may not be adjacent to other sites on both sides.

[0055] Use of methylation markers The methylation status of these adjacent CpG sites on a DNA fragment can be used according to the present method to identify the cell type of the cell from which the DNA fragment was derived. 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 that is 200 bp in length and contains 10 CpG sites, its methylation status in NK cells can be expressed as 20% if two of the CpG sites are methylated and eight of them are unmethylated. If the average methylation status of F1 in all other cell types, i.e., cell types that do not contain NK cells, is in the range of 70% to 90%, F1 can be a suitable marker for identifying NK cells. For example, cell-free DNA containing F1 in which two of the 10 CpG sites within F1 are methylated can be determined to have been released from NK cells according to the present method.

[0056] In some embodiments, a methylation percentage cutoff value can be used when determining cell type. Such a cutoff value can be determined based on experimental data, such as those shown in the accompanying experimental examples, using appropriate statistics and can be applied according to the present method. For example, if the methylation percentage of F1 in all tested NK cells ranges from 0 to 40% and in all tested non-NK cells ranges from 60% to 100%, 50% can be applied as a suitable cutoff value. It should be understood that a cutoff value is not necessarily required. For example, if the methylation status of an F1 fragment from an unknown cell is detected and shows 30% methylation, the 30% number can be compared with F1 from NK cells and non-NK cells, and nearest neighbor analysis can be applied to determine the type of the unknown cell.

[0057] In some embodiments, the methylation status of multiple DNA fragments can be collectively used to determine the cell type in a multivariate analysis format. For example, when analyzing cancer cells of unknown primary origin, the methylation status of DNA fragments F1, F2, and F3 can be detected. Multiple methylation percentages can be used to determine the primary cell type of cancer cells using methods such as, but not limited to, random forests, linear regression, support vector machines, and nearest neighbors.

[0058] Disease Detection and Treatment Monitoring Cell typing has important clinical applications. For example, in many diseases, DNA from dying cells is shed into the bloodstream or other bodily fluids (e.g., semen, milk, urine, saliva, and cerebrospinal fluid). Tools that can identify the tissue source of this DNA are useful in identifying and localizing disease. Similarly, changes in the amount of such released DNA can indicate disease progression or treatment efficacy. For example, the method includes measuring the amount of such released DNA at multiple time points, e.g., a first time point and a second time point after the first time point. In some versions, measurements are also made at a third time point after the second time point, and / or at subsequent consecutive time points. In some versions, the second or additional such time point is after treatment for the disease, e.g., cancer, has been administered to the subject, e.g., after resective surgery and / or therapeutic intervention, and / or the first time point is before such treatment. The method can include determining that a disease, e.g., cancer, is worsening or improving based on the difference in DNA amount between two or more, e.g., three or more, four or more, five or more, or ten or more time points. For example, an increase in the amount of disease, e.g., cancer DNA can indicate a worsening disease, e.g., cancer, condition, while a decrease in such DNA can indicate an improving condition. Thus, the method can include providing a disease diagnosis and / or treatment protocol based on the determined difference between the multiple measurements.

[0059] Also, in the case of cancer of unknown primary origin (CUP), identification of the cell type may help identify its origin, which may be key to providing an early disease diagnosis and / or identifying a suitable treatment.

[0060] The methods may include, but are not limited to, detecting carcinoma, lymphoma, blastoma, sarcoma, and leukemia or tissue of origin of lymphoid malignancies. Specific examples of cancer may include, but are not limited to, liver cancer (e.g., hepatocellular carcinoma (F1CC)), hepatoma, liver cancer, 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 or uterine cancer, salivary gland cancer, kidney 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 squamous cell carcinoma of the lung, peritoneal cancer, gastric (gastric or cancer of the stomach, 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 carcinoma (NPC). Further examples of cancer include, but are not limited to, fibrosarcoma, choriocarcinoma, laryngeal cancer, retinoblastoma, capsular tumor, androgenic tumor, hematologic malignancies (including, but not limited to, non-Hodgkin's lymphoma (NHL), multiple myeloma, and acute hematologic malignancies), endometriosis, Kaposi's sarcoma, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcoma, urinary tract cancer, schwannoma, oligodendroglioma, and neuroblastoma.

[0061] In some embodiments, the cancer according to the present disclosure can be uterine cancer, upper GI squamous cell carcinoma, all other upper GI cancers, thyroid cancer, sarcoma, urothelial renal cancer, all other renal cancers, prostate cancer, pancreatic cancer, ovarian cancer, neuroendocrine cancer, multiple myeloma, melanoma, lymphoma, small cell lung cancer, lung adenocarcinoma, all other lung cancers, leukemia, hepatobiliary cancer, hepatobiliary cancer, head and neck cancer, colorectal cancer, cervical cancer, breast cancer, bladder cancer, anorectal cancer, or any combination thereof. The cancer according to the present embodiments can also be anal cancer, esophageal cancer, head and neck cancer, liver / bile duct cancer, lung cancer, ovarian cancer, pancreatic cancer, plasma cell neoplasm, gastric cancer, or any combination thereof. The cancer according to this embodiment can be thyroid cancer, melanoma, myeloid neoplasm, renal cancer, prostate cancer, breast cancer, uterine cancer, ovarian cancer, bladder cancer, urothelial cancer, cervical cancer, anorectal cancer, head and neck cancer, colorectal cancer, liver cancer, bile duct cancer, pancreatic cancer, gallbladder cancer, upper GI cancer, multiple myeloma, lymphoid neoplasm, lung cancer, or any combination thereof.

[0062] Various examples of clinical applications of this technology are described in further detail below with respect to exemplary cell types and groups of cell types.

[0063] A. Gastrointestinal cells The gastrointestinal (GI) system, or GI tract, is the tube from the mouth to the anus and includes all organs of the digestive system in humans and other animals. Food is taken in through the mouth and digested to extract nutrients and absorb energy, and waste products are excreted as feces. Given their shared functionality, the various different types of cells and tissues in this system share several common molecular features, including genetic and epigenetic characteristics.

[0064] A.1. Oral, laryngeal, and esophageal epithelial cells Some genomic locations are found herein to be uniformly hypomethylated or hypermethylated in oral, laryngeal, and esophageal epithelial cells compared to all other cell types in humans (see, e.g., Table A). For example, the genomic sequences provided in SEQ ID NOS: 1-15, 16-90, 91-91, 92-101, or 102-125 (annotated with the start and end positions on the respective chromosomes) all have a methylation percentage of less than 40% in oral, laryngeal, or esophageal epithelial cells and a methylation percentage of greater than 60% in all other cell types. Similarly, the genomic sequences provided in SEQ ID NOS: 126-133, 134-134, or 135-150 all have a relatively high methylation percentage (greater than 60%) in oral, laryngeal, or esophageal epithelial cells and a low methylation percentage (less than 40%) in all other cell types.

[0065] [Table 1-1]

[0066] [Table 1-2]

[0067] [Table 1-3]

[0068] [Table 1-4]

[0069] [Table 1-5]

[0070] [Table 1-6]

[0071] [Table 1-7] * U: lower methylation (unmethylated) in certain cell types and higher methylation in other cell types; M: Higher methylation (methylation) in certain cell types and lower methylation in other cell types.

[0072] Each genome sequence in the Sequence Listing (according to the human genome version hg19, Genome Reference Consortium Human Build 37 (GRCh37), published February 27, 2009) represents a DNA fragment that includes or overlaps with the genome sequence. In some embodiments, a DNA fragment containing a CpG cluster that can be used as a methylation marker comprises at least one CpG site contained in the genome sequence defined in the Sequence Listing. In some embodiments, the DNA fragment comprises at least two, three, four, five, six, seven, eight, nine, ten, or more CpG sites contained in the genome sequence defined in the Sequence Listing.

[0073] A sequence listing is concurrently submitted in ASCII format and is incorporated herein by reference in its entirety. A listing of all sequences, without the actual sequence, is provided in Table B. Each sequence (see examples in Table C) is annotated with respect to its genomic location (e.g., chr9:119238427-119238709), nearby genes and locations (e.g., introns of ASTN2), and region, 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 within the cell type relative to all other cell types (e.g., 0.05:0.94).

[0074] [Table 2-1]

[0075]

Table 2-2

[0076]

Table 2-3

[0077]

Table 2-4

[0078]

Table 2-5

[0079]

Table 2-6

[0080]

Table 2-7

[0081]

Table 2-8

[0082]

Table 2-9

[0083]

Table 2-10

[0084]

Table 2-11

[0085]

Table 2-12

[0086]

Table 2-13

[0087]

Table 2-14

[0088]

Table 2-15

[0089]

Table 2-16

[0090]

Table 2-17

[0091]

Table 2-18

[0092]

Table 2-19

[0093]

Table 2-20

[0094]

Table 2-21

[0095]

Table 2-22

[0096]

Table 2-23

[0097]

Table 2-24

[0098]

Table 2-25

[0099]

Table 2-26

[0100]

Table 2-27

[0101]

Table 2-28

[0102]

Table 2-29

[0103]

Table 2-30

[0104]

Table 2-31

[0105]

Table 2-32

[0106]

Table 2-33

[0107]

Table 2-34

[0108]

Table 2-35

[0109]

Table 2-36

[0110]

Table 2-37

[0111]

Table 2-38

[0112]

Table 2-39

[0113]

Table 2-40

[0114]

Table 2-41

[0115]

Table 2-42

[0116]

Table 2-43

[0117]

Table 2-44

[0118]

Table 2-45

[0119]

Table 2-46

[0120]

Table 2-47

[0121]

Table 2-48

[0122]

Table 2-49

[0123]

Table 2-50

[0124]

Table 2-51

[0125]

Table 2-52

[0126]

Table 2-53

[0127]

Table 2-54

[0128]

Table 2-55

[0129]

Table 2-56

[0130]

Table 2-57

[0131]

Table 2-58

[0132]

Table 2-59

[0133]

Table 2-60

[0134]

Table 2-61

[0135]

Table 2-62

[0136]

Table 2-63

[0137]

Table 2-64

[0138]

Table 2-65

[0139]

Table 2-66

[0140]

Table 2-67

[0141]

Table 2-68

[0142]

Table 2-69

[0143]

Table 2-70

[0144]

Table 2-71

[0145]

Table 2-72

[0146]

Table 2-73

[0147]

Table 2-74

[0148]

Table 2-75

[0149]

Table 2-76

[0150]

Table 2-77

[0151]

Table 2-78

[0152]

Table 2-79

[0153]

Table 2-80

[0154]

Table 2-81

[0155]

Table 2-82

[0156]

Table 2-83

[0157]

Table 2-84

[0158]

Table 2-85

[0159]

Table 2-86

[0160]

Table 2-87

[0161]

Table 2-88

[0162]

Table 2-89

[0163]

Table 2-90

[0164]

Table 2-91

[0165]

Table 2-92

[0166]

Table 2-93

[0167]

Table 2-94

[0168]

Table 2-95

[0169]

Table 2-96

[0170]

Table 2-97

[0171]

Table 2-98

[0172]

Table 2-99

[0173]

Table 2-100

[0174]

Table 2-101

[0175]

Table 2-102

[0176]

Table 2-103

[0177]

Table 2-104

[0178]

Table 2-105

[0179]

Table 2-106

[0180]

Table 2-107

[0181]

Table 2-108

[0182]

Table 2-109

[0183]

Table 2-110

[0184]

Table 2-111

[0185]

Table 2-112

[0186]

Table 2-113

[0187]

Table 2-114

[0188]

Table 2-115

[0189]

Table 2-116

[0190]

Table 2-117

[0191]

Table 2-118

[0192]

Table 2-119

[0193]

Table 2-120

[0194]

Table 2-121

[0195]

Table 2-122

[0196]

Table 2-123

[0197]

Table 2-124

[0198]

Table 2-125

[0199]

Table 2-126

[0200]

Table 2-127

[0201]

Table 2-128

[0202]

Table 2-129

[0203]

Table 2-130

[0204]

Table 2-131

[0205]

Table 2-132

[0206]

Table 2-133

[0207]

Table 2-134

[0208]

Table 2-135

[0209]

Table 2-136

[0210]

Table 2-137

[0211]

Table 2-138

[0212]

Table 2-139

[0213]

Table 2-140

[0214]

Table 2-141

[0215]

Table 2-142

[0216]

Table 2-143

[0217]

Table 2-144

[0218]

Table 2-145

[0219]

Table 2-146

[0220]

Table 2-147

[0221]

Table 2-148

[0222]

Table 2-149

[0223]

Table 2-150

[0224]

Table 2-151

[0225]

Table 2-152

[0226]

Table 2-153

[0227]

Table 2-154

[0228]

Table 2-155

[0229]

Table 2-156

[0230]

Table 2-157

[0231]

Table 2-158

[0232]

Table 2-159

[0233]

Table 2-160

[0234]

Table 2-161

[0235]

Table 2-162

[0236]

Table 2-163

[0237]

Table 2-164

[0238]

Table 2-165

[0239]

Table 2-166

[0240]

Table 2-167

[0241] Table 2-168

[0242]

Table 2-169

[0243]

Table 2-170

[0244]

Table 2-171

[0245]

Table 2-172

[0246]

Table 2-173

[0247]

Table 2-174

[0248]

Table 2-175

[0249]

Table 2-176

[0250]

Table 2-177

[0251]

Table 2-178

[0252]

Table 2-179

[0253]

Table 2-180

[0254]

Table 2-181

[0255]

Table 2-182

[0256]

Table 2-183

[0257]

Table 2-184

[0258]

Table 2-185

[0259]

Table 2-186

[0260]

Table 2-187

[0261] Table 2-188

[0262]

Table 2-189

[0263]

Table 2-190

[0264]

Table 2-191

[0265]

Table 2-192

[0266]

Table 2-193

[0267]

Table 2-194

[0268]

Table 2-195

[0269]

Table 2-196

[0270]

Table 2-197

[0271] Table 2-198

[0272]

Table 2-199

[0273]

Table 2-200

[0274]

Table 2-201

[0275]

Table 2-202

[0276]

Table 2-203

[0277]

Table 2-204

[0278]

Table 2-205

[0279]

Table 2-206

[0280]

Table 2-207

[0281]

Table 2-208

[0282]

Table 2-209

[0283]

Table 2-210

[0284]

Table 2-211

[0285]

Table 2-212

[0286]

Table 2-213

[0287]

Table 2-214

[0288]

Table 2-215

[0289]

Table 2-216

[0290]

Table 2-217

[0291]

Table 2-218

[0292]

Table 2-219

[0293]

Table 2-220

[0294]

Table 2-221

[0295]

Table 2-222

[0296]

Table 2-223

[0297]

Table 2-224

[0298]

Table 2-225

[0299]

Table 2-226

[0300]

Table 2-227

[0301]

Table 2-228

[0302]

Table 2-229

[0303]

Table 3

[0304] According to one embodiment of the present disclosure, a method is provided for identifying a biological sample as containing DNA from oral, laryngeal, or esophageal epithelial cells. In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 1-15 or 16-90. In some embodiments, the method then identifies the target DNA fragment as being from oral, laryngeal, or esophageal epithelial cells if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as being from an oral, laryngeal, or esophageal epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies a target DNA fragment as being from an oral, laryngeal, or esophageal epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies a target DNA fragment as not being from an oral, laryngeal, or esophageal epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from an oral, laryngeal, or esophageal epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0305] In some embodiments, the method involves detecting the methylation status of a plurality (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) of CpG sites of a target DNA fragment in a biological sample, wherein at least one (or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites are located within or within 100 bp, 200 bp, 500 bp, or 1 kb of a human genomic sequence selected from SEQ ID NOs: 126-133. In some embodiments, the method then identifies the target DNA fragment as being from an oral, laryngeal, or esophageal epithelial cell if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from an oral, laryngeal, or esophageal epithelial cell if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies a target DNA fragment as being from an oral, laryngeal, or esophageal epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies a target DNA fragment as not being from an oral, laryngeal, or esophageal epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as not being from an oral, laryngeal, or esophageal epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0306] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 1-15, 16-90, 91-91, 92-101, 102-125, 126-133, 134-134, or 135-150.

[0307] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 1-15, 16-90, 91-91, 92-101, or 102-125. In some embodiments, the method then identifies the target DNA fragment as being from an oral cavity, laryngeal, or esophageal epithelial cell if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as being from an oral, laryngeal, or esophageal epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies a target DNA fragment as being from an oral, laryngeal, or esophageal epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies a target DNA fragment as not being from an oral, laryngeal, or esophageal epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from an oral, laryngeal, or esophageal epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0308] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites are located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 126-133, 134-134, or 135-150. In some embodiments, the method then identifies the target DNA fragment as being from an oral cavity, laryngeal, or esophageal epithelial cell if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as being from an oral, laryngeal, or esophageal epithelial cell if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies a target DNA fragment as being from an oral, laryngeal, or esophageal epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies a target DNA fragment as not being from an oral, laryngeal, or esophageal epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from an oral, laryngeal, or esophageal epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0309] In some embodiments, if predictions from two or more of the above methods agree with another, the prediction result is further affirmed.

[0310] The cell type identification method can be used to detect diseases or conditions associated with cell types. In one embodiment, if 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 oral, laryngeal, or esophageal epithelial cells, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is injury, inflammation, or cancer of the oral, laryngeal, or esophageal epithelium.

[0311] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., oral, laryngeal, or esophageal epithelial cells, decreases, e.g., is less at the second time point than at the earlier first time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., oral, laryngeal, or esophageal epithelial cells, increases, e.g., is more at the second time point than at the earlier first time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of worsening. In some embodiments, the subject receives treatment between the two or more tests, and thus the test results indicate treatment efficacy.

[0312] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and, as described above, the methylation state can be used to determine the cell as an oral, laryngeal, or esophageal epithelial cell.

[0313] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon.

[0314] Once the primary origin of the cancer is identified, the subject can be treated with a regimen appropriate for that cancer type.

[0315] A2. Gastric epithelium Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in gastric epithelial cells compared to all other cell types in humans.

[0316] According to one embodiment of the present disclosure, a method for identifying a biological sample as containing DNA from a gastric epithelial cell is provided. In some embodiments, the method involves detecting the methylation status of a plurality (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) of CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 151-170, 171-330, 331-335, 336-340, or 341-378, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 151-170 or 171-330. In some embodiments, the method then identifies the target DNA fragment as being from a gastric epithelial cell if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as being from a gastric epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies a target DNA fragment as being from a gastric epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies a target DNA fragment as not being from a gastric epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as not being from a gastric epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0317] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites are located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 379-401, 402-402, or 403-428, or selected from SEQ ID NOs: 379-401. In some embodiments, the method then identifies the target DNA fragment as being from a gastric epithelial cell if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as being from a gastric epithelial cell if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies a target DNA fragment as being from a gastric epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies a target DNA fragment as not being from a gastric epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as not being from a gastric epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0318] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 151-170, 171-330, 331-335, 336-340, 341-378, 379-401, 402-402, or 403-428.

[0319] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, if 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 gastric epithelial cells, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is gastric epithelial damage, inflammation, or cancer.

[0320] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., gastric epithelial cells, decreases, e.g., is less at the second time point than at the earlier first time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., gastric epithelial cells, increases, e.g., is more at the second time point than at the earlier first time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of worsening. In some embodiments, the subject receives treatment between the two or more tests, and thus the test results indicate treatment efficacy.

[0321] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and the methylation state can be used to identify the cell as a gastric epithelial cell, as described above.

[0322] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0323] A3.Small intestinal epithelium Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in small intestinal epithelial cells compared to all other cell types in humans.

[0324] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from a small intestinal epithelial cell is provided. In some embodiments, the method involves detecting the methylation status of a plurality (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) of CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 429-446, 447-527, 528-529, 530-536, or 537-554, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 429-446 or 447-527. In some embodiments, the method then identifies the target DNA fragment as being from a small intestinal epithelial cell if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a small intestinal epithelial cell if 25%, 30%, 35%, 40%, 45%, or 50% or less of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a small intestinal epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a small intestinal epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from a small intestinal epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0325] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 555-564, 565-565, or 566-579, or selected from SEQ ID NOs: 555-564. In some embodiments, the method then identifies the target DNA fragment as being from a small intestinal epithelial cell if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as being from a small intestinal epithelial cell if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies a target DNA fragment as being from a small intestinal epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies a target DNA fragment as not being from a small intestinal epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as not being from a small intestinal epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0326] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 429-446, 447-527, 528-529, 530-536, 537-554, 555-564, 565-565, or 566-579.

[0327] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, if 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 small intestinal epithelial cells, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is small intestinal epithelial damage, inflammation, or cancer.

[0328] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., small intestinal epithelial cells, decreases, e.g., is less at the second time point of measurement than at the earlier first time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., small intestinal epithelial cells, increases, e.g., is more at the second time point of measurement than at the earlier first time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of worsening. In some embodiments, the subject receives treatment between the two or more tests, and thus the test results indicate a treatment effect.

[0329] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and the methylation state can be used to determine the cell as a small intestinal epithelial cell, as described above.

[0330] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0331] A4. Colonic epithelium Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in colonic epithelial cells compared to all other cell types in humans.

[0332] According to one embodiment of the present disclosure, a method is provided for identifying a biological sample as containing DNA from a colon epithelial cell. In some embodiments, the method involves detecting the methylation status of a plurality (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) of CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 580-596, 597-657, 658-660, 661-668, or 669-704, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 580-596 or 597-657. In some embodiments, the method then identifies the target DNA fragment as being from a colon epithelial cell if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a colon epithelial cell if 25%, 30%, 35%, 40%, 45%, or 50% or less of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a colon epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a colon epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not from a colon epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0333] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 705-715 or 716-729, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 705-715. In some embodiments, the method then identifies the target DNA fragment as being from a colon epithelial cell if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as being from a colon epithelial cell if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies a target DNA fragment as being from a colon epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies a target DNA fragment as not being from a colon epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as not being from a colon epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0334] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 580-596, 597-657, 658-660, 661-668, 669-704, 705-715, or 716-729.

[0335] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, if 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 colon epithelial cells, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is colon epithelial damage, inflammation, or cancer.

[0336] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., colonic epithelial cells, decreases, e.g., is less at the second time point than at the earlier first time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., colonic epithelial cells, increases, e.g., is more at the second time point than at the earlier first time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of worsening. In some embodiments, the subject receives treatment between the two or more tests, and thus the test results indicate a treatment effect.

[0337] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and the methylation state can be used to determine the cell as a colon epithelial cell, as described above.

[0338] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0339] A5. Colonic fibroblasts Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in colonic fibroblasts compared to all other cell types in humans.

[0340] According to one embodiment of the present disclosure, a method for identifying a biological sample as containing DNA from a colon fibroblast is provided. In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites are located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 730-732. In some embodiments, the method then identifies the target DNA fragment as being from a colon fibroblast if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as being from a colon fibroblast if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies a target DNA fragment as being from a colon fibroblast if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies a target DNA fragment as not being from a colon fibroblast if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not from a colon fibroblast if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0341] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 733-739 or 740-741, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genomic sequence selected from SEQ ID NOs: 733-739. In some embodiments, the method then identifies the target DNA fragment as being from a colon fibroblast if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as being from a colon fibroblast if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies a target DNA fragment as being from a colon fibroblast if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies a target DNA fragment as not being from a colon fibroblast if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not from a colon fibroblast if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0342] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 730-732, 733-739, or 740-741.

[0343] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, if 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 colon fibroblasts, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is colon fibroblast damage, inflammation, or cancer.

[0344] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., colonic fibroblasts, decreases, e.g., is less at the second time point than at the earlier first time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., colonic fibroblasts, increases, e.g., is more at the second time point than at the earlier first time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of worsening. In some embodiments, the subject receives treatment between the two or more tests, and thus the test results indicate a treatment effect.

[0345] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and the methylation state can be used to determine the cell as a colon fibroblast, as described above.

[0346] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0347] A6. Gallbladder epithelium Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in gallbladder epithelial cells compared to all other cell types in humans.

[0348] According to one embodiment of the present disclosure, a method for identifying a biological sample as containing DNA from a gallbladder epithelial cell is provided. In some embodiments, the method involves detecting the methylation status of a plurality (e.g., three, four, five, six, seven, eight, nine, ten or more) of 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 selected from SEQ ID NOs: 742-758, 759-829, 830-831, 832-839, or 840-867, or is located within a human genome sequence selected from SEQ ID NOs: 742-758 or 759-829, or within 100 bp, 200 bp, 500 bp, or 1 kb thereof. In some embodiments, the method then identifies the target DNA fragment as being from a gallbladder epithelial cell if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a gallbladder epithelial cell if 25%, 30%, 35%, 40%, 45%, or 50% or less of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a gallbladder epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a gallbladder epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from a gallbladder epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0349] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 868-875 or 876-876, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 868-875. In some embodiments, the method then identifies the target DNA fragment as being from a gallbladder epithelial cell if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as being from a gallbladder epithelial cell if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies a target DNA fragment as being from a gallbladder epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies a target DNA fragment as not being from a gallbladder epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as not being from a gallbladder epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0350] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 742-758, 759-829, 830-831, 832-839, 840-867, 868-875, or 876-876.

[0351] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, if 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 gallbladder epithelial cells, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is gallbladder epithelial damage, inflammation, or cancer.

[0352] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., gallbladder epithelial cells, decreases, e.g., is less at the second time point than at the earlier first time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., gallbladder epithelial cells, increases, e.g., is more at the second time point than at the earlier first time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of worsening. In some embodiments, the subject receives treatment between the two or more tests, and thus the test results indicate treatment efficacy.

[0353] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and the methylation state can be used to identify the cell as a gallbladder epithelial cell, as described above.

[0354] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0355] A7. Liver hepatocytes Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in liver hepatocytes compared to all other cell types in humans.

[0356] 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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 877-896, 897-980, 981-983, 984-986, 987-988, or 989-1002, or is located within a human genome sequence selected from SEQ ID NOs: 877-896 or 897-980, or within 100 bp, 200 bp, 500 bp, or 1 kb thereof. In some embodiments, the method then identifies the target DNA fragment as being from a liver hepatocyte if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a liver hepatocyte if 25%, 30%, 35%, 40%, 45%, or 50% or less of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a liver hepatocyte if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a liver hepatocyte if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from a liver hepatocyte if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0357] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 1003-1018, 1019-1023, or 1024-1027, or is located within or within 100 bp, 200 bp, 500 bp, or 1 kb of a human genome sequence selected from SEQ ID NOs: 1003-1018. In some embodiments, the method then identifies the target DNA fragment as being from a liver hepatocyte if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a liver hepatocyte if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a liver hepatocyte if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a liver hepatocyte if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from a liver hepatocyte if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0358] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 877-896, 897-980, 981-983, 984-986, 987-988, 989-1002, 1003-1018, 1019-1023, or 1024-1027.

[0359] The cell type identification method can be used to detect diseases or conditions associated with cell types. In one embodiment, if 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 liver hepatocytes, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is liver hepatocyte damage, inflammation, or cancer.

[0360] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., liver hepatocytes, decreases, e.g., is less at the second time point of measurement than at the first, earlier time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., liver hepatocytes, increases, e.g., is more at the second time point of measurement than at the first, earlier time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of worsening. In some embodiments, the subject receives treatment between the two or more tests, and thus the test results indicate treatment efficacy.

[0361] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and the methylation state can be used to identify the cell as a liver hepatocyte, as described above.

[0362] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0363] A8. Pancreatic acinar cells Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in pancreatic acinar cells compared to all other cell types in humans.

[0364] According to one embodiment of the present disclosure, a method for identifying a biological sample as containing DNA from pancreatic acinar cells is provided. In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 1028-1041, 1042-1112, 1113-1116, 1117-1127, or 1128-1155, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 1028-1041 or 1042-1112. In some embodiments, the method then identifies the target DNA fragment as being from a pancreatic acinar cell if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a pancreatic acinar cell if 25%, 30%, 35%, 40%, 45%, or 50% or less of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a pancreatic acinar cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a pancreatic acinar cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from a pancreatic acinar cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0365] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 1156-1161 or 1162-1180, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 1156-1161. In some embodiments, the method then identifies the target DNA fragment as being from a pancreatic acinar cell if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as being from a pancreatic acinar cell if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies a target DNA fragment as being from a pancreatic acinar cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies a target DNA fragment as not being from a pancreatic acinar cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as not being from a pancreatic acinar cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0366] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 1028-1041, 1042-1112, 1113-1116, 1117-1127, 1128-1155, 1156-1161, or 1162-1180.

[0367] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, if 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 the subject, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is pancreatic acinar cell injury, inflammation, or cancer. In some embodiments, the disease is diabetes.

[0368] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., pancreatic acinar cells, decreases, e.g., is less at the second time point of measurement than at the first, earlier time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., pancreatic acinar cells, increases, e.g., is more at the second time point of measurement than at the first, earlier time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of worsening. In some embodiments, the subject receives treatment between the two or more tests, and thus the test results indicate treatment efficacy.

[0369] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and the methylation state can be used to determine the cell as a pancreatic acinar cell, as described above.

[0370] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0371] A9. Pancreatic alpha cells Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in pancreatic alpha cells compared to all other cell types in humans.

[0372] According to one embodiment of the present disclosure, a method for identifying a biological sample as containing DNA from pancreatic alpha cells is provided. In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 1181-1198, 1199-1282, 1283-1284, 1285-1287, 1288-1292, or 1293-1306, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 1181-1198 or 1199-1282. In some embodiments, the method then identifies the target DNA fragment as being from a pancreatic alpha cell if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a pancreatic alpha cell if 25%, 30%, 35%, 40%, 45%, or 50% or less of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a pancreatic alpha cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a pancreatic alpha cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from a pancreatic alpha cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0373] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites are located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 1307-1315, 1316-1316, or 1317-1331, or selected from SEQ ID NOs: 1307-1315. In some embodiments, the method then identifies the target DNA fragment as being from a pancreatic alpha cell if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as being from a pancreatic alpha cell if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies a target DNA fragment as being from a pancreatic alpha cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies a target DNA fragment as not being from a pancreatic alpha cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as not being from a pancreatic alpha cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0374] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 1181-1198, 1199-1282, 1283-1284, 1285-1287, 1288-1292, 1293-1306, 1307-1315, 1316-1316, or 1317-1331.

[0375] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, if 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 subject's pancreatic alpha cells, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is pancreatic alpha cell damage, inflammation, or cancer. In some embodiments, the disease is diabetes.

[0376] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., pancreatic alpha cells, decreases, e.g., is less at the second time point than at the earlier first time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., pancreatic alpha cells, increases, e.g., is more at the second time point than at the earlier first time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of worsening. In some embodiments, the subject receives treatment between the two or more tests, and thus the test results indicate treatment efficacy.

[0377] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and the methylation state can be used to identify the cell as a pancreatic alpha cell, as described above.

[0378] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0379] A10. Pancreatic beta cells Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in pancreatic beta cells compared to all other cell types in humans.

[0380] According to one embodiment of the present disclosure, a method for identifying a biological sample as containing DNA from pancreatic beta cells is provided. In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 1332-1351, 1352-1440, 1441-1445, or 1446-1460, or is located within or within 100 bp, 200 bp, 500 bp, or 1 kb of a human genome sequence selected from SEQ ID NOs: 1332-1351 or 1352-1440. In some embodiments, the method then identifies the target DNA fragment as being from a pancreatic beta cell if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a pancreatic beta cell if 25%, 30%, 35%, 40%, 45%, or 50% or less of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a pancreatic beta cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a pancreatic beta cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from a pancreatic beta cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0381] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 1461-1471 or 1472-1485, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 1461-1471. In some embodiments, the method then identifies the target DNA fragment as being from a pancreatic beta cell if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as being from a pancreatic beta cell if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies a target DNA fragment as being from a pancreatic beta cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies a target DNA fragment as not being from a pancreatic beta cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as not being from a pancreatic beta cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0382] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 1332-1351, 1352-1440, 1441-1445, 1446-1460, 1461-1471, or 1472-1485.

[0383] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, if 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 pancreatic beta cells, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is pancreatic beta cell damage, inflammation, or cancer. In some embodiments, the disease is diabetes.

[0384] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., pancreatic beta cells, decreases, e.g., is less at the second time point of measurement than at the first, earlier time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., pancreatic beta cells, increases, e.g., is more at the second time point of measurement than at the first, earlier time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of worsening. In some embodiments, the subject receives treatment between the two or more tests, and thus the test results indicate treatment efficacy.

[0385] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and the methylation state can be used to identify the cell as a pancreatic beta cell, as described above.

[0386] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0387] A11. Pancreatic delta cells Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in pancreatic delta cells compared to all other cell types in humans.

[0388] According to one embodiment of the present disclosure, a method for identifying a biological sample as containing DNA from a pancreatic delta cell is provided. In some embodiments, the method involves detecting the methylation status of a plurality (e.g., three, four, five, six, seven, eight, nine, ten or more) of 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 selected from SEQ ID NOs: 1486-1508, 1509-1594, 1595-1596, 1597-1598, or 1599-1613, or is located within a human genome sequence selected from SEQ ID NOs: 1486-1508 or 1509-1594, or within 100 bp, 200 bp, 500 bp, or 1 kb thereof. In some embodiments, the method then identifies the target DNA fragment as being from a pancreatic delta cell if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a pancreatic delta cell if 25%, 30%, 35%, 40%, 45%, or 50% or less of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a pancreatic delta cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a pancreatic delta cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from a pancreatic delta cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0389] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites are selected from SEQ ID NOs: 1614-1624, 1625-1625, or 1626-1638, or are located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 1614-1624. In some embodiments, the method then identifies the target DNA fragment as being from a pancreatic delta cell if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as being from a pancreatic delta cell if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies a target DNA fragment as being from a pancreatic delta cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies a target DNA fragment as not being from a pancreatic delta cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as not being from a pancreatic delta cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0390] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 1486-1508, 1509-1594, 1595-1596, 1597-1598, 1599-1613, 1614-1624, 1625-1625, or 1626-1638.

[0391] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, if 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 pancreatic delta cells, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is pancreatic delta cell damage, inflammation, or cancer. In some embodiments, the disease is diabetes.

[0392] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., pancreatic delta cells, decreases, e.g., is less at the second time point than at the earlier first time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., pancreatic delta cells, increases, e.g., is more at the second time point than at the earlier first time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of worsening. In some embodiments, the subject receives treatment between the two or more tests, and thus the test results indicate treatment efficacy.

[0393] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and the methylation state can be used to identify the cell as a pancreatic delta cell, as described above.

[0394] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0395] A12. Pancreatic duct cells Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in pancreatic duct cells compared to all other cell types in humans.

[0396] According to one embodiment of the present disclosure, a method for identifying a biological sample as containing DNA from pancreatic duct cells is provided. In some embodiments, the method involves detecting the methylation status of a plurality (e.g., three, four, five, six, seven, eight, nine, ten or more) of 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 selected from SEQ ID NOs: 1639-1658, 1659-1742, 1743-1743, 1744-1747, 1748-1751, or 1752-1767, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 1639-1658 or 1659-1742. In some embodiments, the method then identifies the target DNA fragment as being from a pancreatic duct cell if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a pancreatic duct cell if 25%, 30%, 35%, 40%, 45%, or 50% or less of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a pancreatic duct cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a pancreatic duct cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from a pancreatic duct cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0397] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 1768-1779 or 1780-1792, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 1768-1779. In some embodiments, the method then identifies the target DNA fragment as being from a pancreatic duct cell if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as being from a pancreatic duct cell if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies a target DNA fragment as being from a pancreatic duct cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies a target DNA fragment as not being from a pancreatic duct cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as not being from a pancreatic duct cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0398] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 1639-1658, 1659-1742, 1743-1743, 1744-1747, 1748-1751, 1752-1767, 1768-1779, or 1780-1792.

[0399] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, if 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 duct cells of the subject, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is pancreatic duct cell injury, inflammation, or cancer. In some embodiments, the disease is diabetes.

[0400] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., pancreatic duct cells, decreases, e.g., is less at the second time point than at the earlier first time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., pancreatic duct cells, increases, e.g., is more at the second time point than at the earlier first time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of worsening. In some embodiments, the subject receives treatment between the two or more tests, and thus the test results indicate treatment efficacy.

[0401] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and the methylation state can be used to determine the cell as a pancreatic duct cell, as described above.

[0402] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0403] Group 1-GI epithelium (colon epithelium & gastric epithelium & small intestinal epithelium) Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in cell populations, namely colonic epithelium and gastric and small intestinal epithelium, compared to all other cell types in humans.

[0404] According to one embodiment of the present disclosure, a method is provided for identifying that a biological sample contains DNA from a cell selected from colon epithelium, gastric epithelium, and small intestinal epithelium. In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 6541-6556, 6557-6557, or 6558-6565, or is located within or within 100 bp, 200 bp, 500 bp, or 1 kb of a human genome sequence selected from SEQ ID NOs: 6541-6556. In some embodiments, the method then identifies the target DNA fragment as being from a cell selected from colonic epithelium, gastric epithelium, and small intestinal epithelium if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a cell selected from colonic epithelium, gastric epithelium, and small intestinal epithelium if 25%, 30%, 35%, 40%, 45%, or 50% or less of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a cell selected from colonic epithelium, gastric epithelium, and small intestinal epithelium if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not from a cell selected from colonic epithelium, gastric epithelium, and small intestinal epithelium if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not from a cell selected from colonic epithelium, gastric epithelium, and small intestinal epithelium if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0405] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 6541-6556, 6557-6557, or 6558-6565.

[0406] The cell type identification method can be used to detect diseases or conditions associated with cell types. In one embodiment, if 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 the subject, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. 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.

[0407] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., cells selected from colonic epithelium, gastric epithelium, and small intestinal epithelium, decreases, e.g., is less at the second time point than at the earlier first time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., cells selected from colonic epithelium, gastric epithelium, and small intestinal epithelium, increases, e.g., is more at the second time point than at the earlier first time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of worsening. In some embodiments, the subject receives treatment between two or more tests, and thus the test results indicate a treatment effect.

[0408] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments of the cancer cell, and, as described above, the methylation state can be used to determine that the cell is a cell selected from colon epithelium, gastric epithelium, and small intestine epithelium.

[0409] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0410] Group 2 - small intestinal and colonic epithelium Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in cell populations, namely the small intestinal epithelium and colonic epithelium, compared to all other cell types in humans.

[0411] According to one embodiment of the present disclosure, a method is provided for identifying that a biological sample contains DNA from a cell selected from small intestinal epithelium and colonic epithelium. In some embodiments, the method involves detecting the methylation status of a plurality (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) of CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 6695-6702, 6703-6760, 6761-6777, or 6778-6820, or is located within or within 100 bp, 200 bp, 500 bp, or 1 kb of a human genome sequence selected from SEQ ID NOs: 6695-6702 or 6703-6760. In some embodiments, the method then identifies the target DNA fragment as being from a cell selected from small intestinal epithelium and colonic epithelium if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a cell selected from small intestinal epithelium and colonic epithelium if 25%, 30%, 35%, 40%, 45%, or 50% or less of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a cell selected from small intestinal epithelium and colonic epithelium if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not from a cell selected from small intestinal epithelium and colonic epithelium if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not from a cell selected from small intestinal epithelium and colonic epithelium if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0412] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 6821-6825 or 6826-6845, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 6821-6825. In some embodiments, the method then identifies the target DNA fragment as being from a cell selected from small intestinal epithelium and colonic epithelium if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a cell selected from small intestinal epithelium and colonic epithelium if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a cell selected from small intestinal epithelium and colonic epithelium if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a cell selected from small intestinal epithelium and colonic epithelium if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not from a cell selected from small intestinal epithelium and colonic epithelium if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0413] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 6695-6702, 6703-6760, 6761-6777, 6778-6820, 6821-6825, or 6826-6845.

[0414] The cell type identification method can be used to detect diseases or conditions associated with cell types. In one embodiment, if 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 subject's small intestinal epithelium and colonic epithelium, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is injury, inflammation, or cancer of cells selected from the small intestinal epithelium and colonic epithelium.

[0415] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., cells selected from the small intestinal epithelium and colonic epithelium, decreases, e.g., is less at the second time point than at the earlier first time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on the signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., cells selected from the small intestinal epithelium and colonic epithelium, increases, e.g., is more at the second time point than at the earlier first time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on the signs of worsening. In some embodiments, the subject receives treatment between two or more tests, and thus the test results indicate a treatment effect.

[0416] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and, as described above, the methylation state can be used to determine that the cell is a cell selected from small intestinal epithelium and colonic epithelium.

[0417] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0418] Group 3 - Gastric and small intestinal epithelium Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in cell populations, namely, gastric epithelium and small intestinal epithelium, compared to all other cell types in humans.

[0419] According to one embodiment of the present disclosure, a method is provided for identifying that a biological sample contains DNA from a cell selected from gastric epithelium and small intestinal epithelium. In some embodiments, the method involves detecting the methylation status of a plurality (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) of CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 6566-6589, 6590-6672, 6673-6673, 6674-6674, or 6675-6690, or is located within or within 100 bp, 200 bp, 500 bp, or 1 kb of a human genome sequence selected from SEQ ID NOs: 6566-6589 or 6590-6672. In some embodiments, the method then identifies the target DNA fragment as being from a cell selected from gastric epithelium and small intestinal epithelium if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a cell selected from gastric epithelium and small intestinal epithelium if 25%, 30%, 35%, 40%, 45%, or 50% or less of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a cell selected from gastric epithelium and small intestinal epithelium if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a cell selected from gastric epithelium and small intestinal epithelium if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from a cell selected from gastric epithelium and small intestinal epithelium if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0420] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites are selected from SEQ ID NOs: 6691 or 6692-6694, or are located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NO: 6691. In some embodiments, the method then identifies the target DNA fragment as being from a cell selected from gastric epithelium and small intestinal epithelium if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a cell selected from gastric epithelium and small intestinal epithelium if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a cell selected from gastric epithelium and small intestinal epithelium if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a cell selected from gastric epithelium and small intestinal epithelium if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not from a cell selected from gastric epithelium and small intestinal epithelium if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0421] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 6566-6589, 6590-6672, 6673-6673, 6674-6674, 6675-6690, 6691, or 6692-6694.

[0422] The cell type identification method can be used to detect diseases or conditions associated with cell types. In one embodiment, if 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 subject's gastric epithelium and small intestinal epithelium, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is injury, inflammation, or cancer of cells selected from the gastric epithelium and small intestinal epithelium.

[0423] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., cells selected from the gastric epithelium and small intestinal epithelium, decreases, e.g., is less at the second time point than at the earlier first time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on the signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., cells selected from the gastric epithelium and small intestinal epithelium, increases, e.g., is more at the second time point than at the earlier first time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on the signs of worsening. In some embodiments, the subject receives treatment between two or more tests, and thus the test results indicate a treatment effect.

[0424] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and, as described above, the methylation state can be used to determine that the cell is a cell selected from gastric epithelium and small intestinal epithelium.

[0425] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0426] Group 4 - Colon fibroblasts and cardiac fibroblasts Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in cell populations, namely colonic and cardiac fibroblasts, compared to all other cell types in humans.

[0427] According to one embodiment of the present disclosure, a method is provided for identifying that a biological sample contains DNA from cells selected from colon fibroblasts and cardiac fibroblasts. In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 6846-6863, 6864-6869, 6870-6872, 6873-6876, or 6877-6878, or is located within or within 100 bp, 200 bp, 500 bp, or 1 kb of a human genome sequence selected from SEQ ID NOs: 6846-6863 or 6864-6869. In some embodiments, the method then identifies the target DNA fragment as being from a cell selected from colonic fibroblasts and cardiac fibroblasts if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a cell selected from colonic fibroblasts and cardiac fibroblasts if 25%, 30%, 35%, 40%, 45%, or 50% or less of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a cell selected from colonic fibroblasts and cardiac fibroblasts if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not from a cell selected from colonic fibroblasts and cardiac fibroblasts if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not from a cell selected from colonic fibroblasts and cardiac fibroblasts if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0428] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 6879-6890 or 6891-6898, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 6879-6890. In some embodiments, the method then identifies the target DNA fragment as being from a cell selected from a colon fibroblast and a cardiac fibroblast if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a cell selected from colonic fibroblasts and cardiac fibroblasts if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a cell selected from colonic fibroblasts and cardiac fibroblasts if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a cell selected from colonic fibroblasts and cardiac fibroblasts if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not from a cell selected from a colon fibroblast and a cardiac fibroblast if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0429] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 6846-6863, 6864-6869, 6870-6872, 6873-6876, 6877-6878, 6879-6890, or 6891-6898.

[0430] The cell type identification method can be used to detect diseases or conditions associated with cell types. In one embodiment, if 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 colonic fibroblasts and cardiac fibroblasts in the subject, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is injury, inflammation, or cancer of cells selected from colonic fibroblasts and cardiac fibroblasts.

[0431] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., cells selected from colonic fibroblasts and cardiac fibroblasts, is decreased, e.g., less at the second time point than at the earlier first time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on the signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., cells selected from colonic fibroblasts and cardiac fibroblasts, is increased, e.g., more at the second time point than at the earlier first time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on the signs of worsening. In some embodiments, the subject receives treatment between two or more tests, and thus the test results indicate a treatment effect.

[0432] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and, as described above, the methylation state can be used to determine the cell as being selected from a colon fibroblast and a cardiac fibroblast.

[0433] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0434] Group 5 - Pancreatic alpha, beta and delta cells Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in cell populations, namely pancreatic alpha, beta, and delta cells, compared to all other cell types in humans.

[0435] According to one embodiment of the present disclosure, a method is provided for identifying that a biological sample contains DNA from a cell selected from pancreatic alpha, beta, and delta cells. In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 5924-5935, 5936-6011, 6012-6012, 6013-6014, 6015-6026, or 6027-6050, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 5924-5935 or 5936-6011. In some embodiments, the method then identifies the target DNA fragment as being from a cell selected from pancreatic alpha, beta, and delta cells if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a cell selected from pancreatic alpha, beta, and delta cells if 25%, 30%, 35%, 40%, 45%, or 50% or less of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a cell selected from pancreatic alpha, beta, and delta cells if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a cell selected from pancreatic alpha, beta, and delta cells if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from a cell selected from pancreatic alpha, beta, and delta cells if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0436] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 6051-6057 or 6058-6075, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 6051-6057. In some embodiments, the method then identifies the target DNA fragment as being from a cell selected from pancreatic alpha, beta, and delta cells if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a cell selected from pancreatic alpha, beta, and delta cells if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a cell selected from pancreatic alpha, beta, and delta cells if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a cell selected from pancreatic alpha, beta, and delta cells if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from a cell selected from pancreatic alpha, beta, and delta cells if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0437] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 5924-5935, 5936-6011, 6012-6012, 6013-6014, 6015-6026, 6027-6050, 6051-6057, or 6058-6075.

[0438] The cell type identification method can be used to detect diseases or conditions associated with cell types. In one embodiment, if 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 pancreatic alpha, beta, and delta cells of the subject, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is damage, inflammation, or cancer of cells selected from pancreatic alpha, beta, and delta cells.

[0439] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., cells selected from pancreatic alpha, beta, and delta cells, decreases, e.g., is less at the second time point than at the earlier first time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., cells selected from pancreatic alpha, beta, and delta cells, increases, e.g., is more at the second time point than at the earlier first time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of worsening. In some embodiments, the subject receives treatment between two or more tests, and thus the test results indicate a treatment effect.

[0440] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and, as described above, the methylation state can be used to determine the cell as a cell selected from pancreatic alpha, beta, and delta cells.

[0441] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0442] B. Genitourinary cells B1. Endometrial epithelium Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in endometrial epithelial cells compared to all other cell types in humans.

[0443] According to one embodiment of the present disclosure, a method for identifying a biological sample as containing DNA from an endometrial epithelial cell is provided. In some embodiments, the method involves detecting the methylation status of a plurality (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) of CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 1793-1864, 1865-1872, or 1873-1892, or selected from SEQ ID NOs: 1793-1864. In some embodiments, the method then identifies the target DNA fragment as being from an endometrial epithelial cell if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as being from an endometrial epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies a target DNA fragment as being from an endometrial epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies a target DNA fragment as not being from an endometrial epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from an endometrial epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0444] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 1893-1905 or 1906-1917, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 1893-1905. In some embodiments, the method then identifies the target DNA fragment as being from an endometrial epithelial cell if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as being from an endometrial epithelial cell if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies a target DNA fragment as being from an endometrial epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies a target DNA fragment as not being from an endometrial epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from an endometrial epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0445] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 1793-1864, 1865-1872, 1873-1892, 1893-1905, or 1906-1917.

[0446] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, if 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 subject's endometrial epithelial cells, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is endometrial epithelial damage, inflammation, or cancer.

[0447] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., endometrial epithelial cells, decreases, e.g., is less at the second time point than at the earlier first time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., endometrial epithelial cells, increases, e.g., is more at the second time point than at the earlier first time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of worsening. In some embodiments, the subject receives treatment between two or more tests, and thus the test results indicate a treatment effect.

[0448] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and the methylation state can be used to identify the cell as an endometrial epithelial cell, as described above.

[0449] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0450] B2. Fallopian epithelium Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in fallopian epithelial cells compared to all other cell types in humans.

[0451] According to one embodiment of the present disclosure, a method for identifying a biological sample as containing DNA from a fallopian epithelial cell is provided. In some embodiments, the method involves detecting the methylation status of a plurality (e.g., three, four, five, six, seven, eight, nine, ten or more) of 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 selected from SEQ ID NOs: 1918-1937, 1938-2022, 2023-2024, 2025-2029, or 2030-2042, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 1918-1937 or 1938-2022. In some embodiments, the method then identifies the target DNA fragment as being from a fallopian epithelial cell if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a fallopian epithelial cell if 25%, 30%, 35%, 40%, 45%, or 50% or less of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a fallopian epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a fallopian epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not from a fallopian epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0452] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 2043-2061 or 2062-2067, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 2043-2061. In some embodiments, the method then identifies the target DNA fragment as being from a fallopian epithelial cell if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as being from a fallopian epithelial cell if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies a target DNA fragment as being from a fallopian epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies a target DNA fragment as not being from a fallopian epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not from a fallopian epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0453] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 1918-1937, 1938-2022, 2023-2024, 2025-2029, 2030-2042, 2043-2061, or 2062-2067.

[0454] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, if 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 fallopian epithelial cells, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is fallopian epithelial damage, inflammation, or cancer.

[0455] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., fallopian epithelial cells, decreases, e.g., is less at the second time point of measurement than at the first, earlier time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on the signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., fallopian epithelial cells, increases, e.g., is more at the second time point of measurement than at the first, earlier time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on the signs of worsening. In some embodiments, the subject receives treatment between two or more tests, and thus the test results indicate a treatment effect.

[0456] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and the methylation state can be used to determine the cell as a fallopian epithelial cell, as described above.

[0457] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0458] B3. Renal epithelium Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in kidney epithelial cells compared to all other cell types in humans.

[0459] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from a kidney epithelial cell is provided. In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 2068-2080, 2081-2141, 2142-2144, 2145-2156, or 2157-2194, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 2068-2080 or 2081-2141. In some embodiments, the method then identifies the target DNA fragment as being from a kidney epithelial cell if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a kidney epithelial cell if 25%, 30%, 35%, 40%, 45%, or 50% or less of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a kidney epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a kidney epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from a kidney epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0460] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 2195-2209 or 2210-2219, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 2195-2209. In some embodiments, the method then identifies the target DNA fragment as being from a kidney epithelial cell if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as being from a kidney epithelial cell if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies a target DNA fragment as being from a kidney epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies a target DNA fragment as not being from a kidney epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as not being from a kidney epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0461] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 2068-2080, 2081-2141, 2142-2144, 2145-2156, 2157-2194, 2195-2209, or 2210-2219.

[0462] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, if 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 renal epithelial cells, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is renal epithelial injury, inflammation, or cancer.

[0463] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., kidney epithelial cells, decreases, e.g., is less at the second time point than at the earlier first time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., kidney epithelial cells, increases, e.g., is more at the second time point than at the earlier first time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of worsening. In some embodiments, the subject receives treatment between two or more tests, and thus the test results indicate a treatment effect.

[0464] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and the methylation state can be used to identify the cell as a renal epithelial cell, as described above.

[0465] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0466] B4. Bladder epithelium Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in bladder epithelial cells compared to all other cell types in humans.

[0467] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from a bladder epithelial cell is provided. In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 2220-2233, 2234-2298, 2299-2299, 2300-2303, 2304-2313, or 2314-2345, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 2220-2233 or 2234-2298. In some embodiments, the method then identifies the target DNA fragment as being from a bladder epithelial cell if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a bladder epithelial cell if 25%, 30%, 35%, 40%, 45%, or 50% or less of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a bladder epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a bladder epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from a bladder epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0468] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 2346-2350, 2351-2351, or 2352-2370, or is located within or within 100 bp, 200 bp, 500 bp, or 1 kb of a human genome sequence selected from SEQ ID NOs: 2346-2350. In some embodiments, the method then identifies the target DNA fragment as being from a bladder epithelial cell if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as being from a bladder epithelial cell if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies a target DNA fragment as being from a bladder epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies a target DNA fragment as not being from a bladder epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as not being from a bladder epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0469] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 2220-2233, 2234-2298, 2299-2299, 2300-2303, 2304-2313, 2314-2345, 2346-2350, 2351-2351, or 2352-2370.

[0470] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, if 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 subject's bladder epithelial cells, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is bladder epithelial damage, inflammation, or cancer.

[0471] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., bladder epithelial cells, decreases, e.g., is less at the second time point than at the earlier first time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., bladder epithelial cells, increases, e.g., is more at the second time point than at the earlier first time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of worsening. In some embodiments, the subject receives treatment between two or more tests, and thus the test results indicate a treatment effect.

[0472] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and the methylation state can be used to identify the cell as a bladder epithelial cell, as described above.

[0473] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0474] B5. Prostate epithelium Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in prostate epithelial cells compared to all other cell types in humans.

[0475] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from a prostate epithelial cell is provided. In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 2371-2389, 2390-2476, 2477-2480, 2481-2486, or 2487-2495, or is located within a human genome sequence selected from SEQ ID NOs: 2371-2389 or 2390-2476, or within 100 bp, 200 bp, 500 bp, or 1 kb thereof. In some embodiments, the method then identifies the target DNA fragment as being from a prostate epithelial cell if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a prostate epithelial cell if 25%, 30%, 35%, 40%, 45%, or 50% or less of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a prostate epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a prostate epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from a prostate epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0476] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 2496-2500, 2501-2501, or 2502-2520, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 2496-2500. In some embodiments, the method then identifies the target DNA fragment as being from a prostate epithelial cell if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a prostate epithelial cell if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a prostate epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a prostate epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from a prostate epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0477] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 2371-2389, 2390-2476, 2477-2480, 2481-2486, 2487-2495, 2496-2500, 2501-2501, or 2502-2520.

[0478] The cell type identification method can be used to detect diseases or conditions associated with cell types. In one embodiment, if 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 prostate epithelial cells, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is prostate epithelial damage, inflammation, or cancer.

[0479] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., prostate epithelial cells, decreases, e.g., is less at the second time point than at the earlier first time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., prostate epithelial cells, increases, e.g., is more at the second time point than at the earlier first time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of worsening. In some embodiments, the subject receives treatment between two or more tests, and thus the test results indicate a treatment effect.

[0480] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and the methylation state can be used to determine the cell as a prostate epithelial cell, as described above.

[0481] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0482] B6. Breast basal epithelium Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in breast basal epithelial cells compared to all other cell types in humans.

[0483] According to one embodiment of the present disclosure, a method for identifying a biological sample as containing DNA from a breast basal epithelial cell is provided. In some embodiments, the method involves detecting the methylation status of a plurality (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) of CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 2521-2536, 2537-2616, 2617-2625, or 2626-2651, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 2521-2536 or 2537-2616. In some embodiments, the method then identifies the target DNA fragment as being from a breast basal epithelial cell if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a breast basal epithelial cell if 25%, 30%, 35%, 40%, 45%, or 50% or less of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a breast basal epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a breast basal epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from a breast basal epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0484] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 2652-2659 or 2660-2676, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 2652-2659. In some embodiments, the method then identifies the target DNA fragment as being from a breast basal epithelial cell if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a breast basal epithelial cell if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a breast basal epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a breast basal epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from a breast basal epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0485] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 2521-2536, 2537-2616, 2617-2625, 2626-2651, 2652-2659, or 2660-2676.

[0486] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, if 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 breast basal epithelial cells, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is breast basal epithelial damage, inflammation, or cancer.

[0487] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., breast basal epithelial cells, decreases, e.g., is less at the second time point than at the earlier first time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on the signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., breast basal epithelial cells, increases, e.g., is more at the second time point than at the earlier first time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on the signs of worsening. In some embodiments, the subject receives treatment between two or more tests, and thus the test results indicate a treatment effect.

[0488] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and the methylation state can be used to determine the cell as a breast basal epithelial cell, as described above.

[0489] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0490] B7. Breast luminal epithelium Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in breast luminal epithelial cells compared to all other cell types in humans.

[0491] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from breast luminal epithelial cells is provided. In some embodiments, the method involves detecting the methylation status of a plurality (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) of CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 2677-2688, 2689-2748, 2749-2749, 2750-2762, or 2763-2802, or is located within a human genome sequence selected from SEQ ID NOs: 2677-2688 or 2689-2748, or within 100 bp, 200 bp, 500 bp, or 1 kb thereof. In some embodiments, the method then identifies the target DNA fragment as being from a breast luminal epithelial cell if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a breast luminal epithelial cell if 25%, 30%, 35%, 40%, 45%, or 50% or less of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a breast luminal epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a breast luminal epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from a breast luminal epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0492] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites are located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genomic sequence selected from SEQ ID NOs: 2803-2815, 2816-2816, or 2817-2827, or selected from SEQ ID NOs: 2803-2815. In some embodiments, the method then identifies the target DNA fragment as being from a breast luminal epithelial cell if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as being from a breast luminal epithelial cell if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies a target DNA fragment as being from a breast luminal epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies a target DNA fragment as not being from a breast luminal epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from a breast luminal epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0493] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 2677-2688, 2689-2748, 2749-2749, 2750-2762, 2763-2802, 2803-2815, 2816-2816, or 2817-2827.

[0494] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, if cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva, or cerebrospinal fluid) is identified as being from breast luminal epithelial cells of the subject, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is breast luminal epithelial damage, inflammation, or cancer.

[0495] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., breast luminal epithelial cells, decreases, e.g., is less at the second time point than at the earlier first time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on the signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., breast luminal epithelial cells, increases, e.g., is more at the second time point than at the earlier first time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on the signs of worsening. In some embodiments, the subject receives treatment between two or more tests, and thus the test results indicate a treatment effect.

[0496] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and the methylation state can be used to determine the cell as a breast luminal epithelial cell, as described above.

[0497] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0498] Group 6 - breast basal epithelium and breast luminal epithelium Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in cell populations, namely, breast basal epithelium and breast luminal epithelium, compared to all other cell types in humans.

[0499] According to one embodiment of the present disclosure, a method is provided for identifying that a biological sample contains DNA from cells selected from breast basal epithelium and breast luminal epithelium. In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 6076-6090, 6091-6159, 6160-6160, 6161-6162, 6163-6171, or 6172-6201, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 6076-6090 or 6091-6159. In some embodiments, the method then identifies the target DNA fragment as being from a cell selected from breast basal epithelium and breast luminal epithelium if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a cell selected from breast basal epithelium and breast luminal epithelium if 25%, 30%, 35%, 40%, 45%, or 50% or less of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a cell selected from breast basal epithelium and breast luminal epithelium if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not from a cell selected from breast basal epithelium and breast luminal epithelium if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not from a cell selected from breast basal epithelium and breast luminal epithelium if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0500] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 6202-6206 or 6207-6226, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genomic sequence selected from SEQ ID NOs: 6202-6206. In some embodiments, the method then identifies the target DNA fragment as being from a cell selected from breast basal epithelium and breast luminal epithelium if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a cell selected from breast basal epithelium and breast luminal epithelium if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a cell selected from breast basal epithelium and breast luminal epithelium if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a cell selected from breast basal epithelium and breast luminal epithelium if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from a cell selected from breast basal epithelium and breast luminal epithelium if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0501] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 6076-6090, 6091-6159, 6160-6160, 6161-6162, 6163-6171, 6172-6201, 6202-6206, or 6207-6226.

[0502] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, if 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 breast basal epithelium and breast luminal epithelium of the subject, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is damage, inflammation, or cancer of cells selected from breast basal epithelium and breast luminal epithelium.

[0503] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., cells selected from breast basal epithelium and breast luminal epithelium, decreases, e.g., is less at the second time point than at the first, earlier time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on the signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., cells selected from breast basal epithelium and breast luminal epithelium, increases, e.g., is more at the second time point than at the first, earlier time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on the signs of worsening. In some embodiments, the subject receives treatment between two or more tests, and thus the test results indicate a treatment effect.

[0504] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments of the cancer cell, and, as described above, the methylation state can be used to determine the cell as being selected from breast basal epithelium and breast luminal epithelium.

[0505] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0506] Group 7 - Fallopian epithelium and ovarian and endometrial epithelium Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in cell groups, namely, fallopian epithelium and ovarian and endometrial epithelium, compared to all other cell types in humans.

[0507] According to one embodiment of the present disclosure, a method is provided for identifying that a biological sample contains DNA from a cell selected from fallopian epithelium, ovarian epithelium, and endometrial epithelium. In some embodiments, the method involves detecting the methylation status of a plurality (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) of CpG sites in a target DNA fragment in the biological sample, wherein at least one (or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 6366-6399, 6400-6468, 6469-6475, 6476-6491, or 6492-6515, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 6366-6399 or 6400-6468. In some embodiments, the method then identifies the target DNA fragment as being from a cell selected from fallopian epithelium, ovarian epithelium, and endometrial epithelium if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a cell selected from fallopian epithelium, ovarian epithelium, and endometrial epithelium if 25%, 30%, 35%, 40%, 45%, or 50% or less of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a cell selected from fallopian epithelium, ovarian epithelium, and endometrial epithelium if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not from a cell selected from fallopian epithelium, ovarian epithelium, and endometrial epithelium if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not from a cell selected from fallopian epithelium, ovarian epithelium, and endometrial epithelium if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0508] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 6516-6527 or 6528-6540, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 6516-6527. In some embodiments, the method then identifies the target DNA fragment as being from a cell selected from fallopian epithelium, ovarian epithelium, and endometrial epithelium if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a cell selected from fallopian epithelium, ovarian epithelium, and endometrial epithelium if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a cell selected from fallopian epithelium, ovarian epithelium, and endometrial epithelium if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a cell selected from fallopian epithelium, ovarian epithelium, and endometrial epithelium if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not from a cell selected from fallopian epithelium and ovarian epithelium and endometrial epithelium if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0509] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 6366-6399, 6400-6468, 6469-6475, 6476-6491, 6492-6515, 6516-6527, or 6528-6540.

[0510] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, if 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 subject's fallopian epithelium, ovarian epithelium, and endometrial epithelium, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is damage, inflammation, or cancer of cells selected from the fallopian epithelium, ovarian epithelium, and endometrial epithelium.

[0511] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., cells selected from fallopian epithelium, ovarian epithelium, and endometrial epithelium, decreases, e.g., is less at the second time point than at the earlier first time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on the signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., cells selected from fallopian epithelium, ovarian epithelium, and endometrial epithelium, increases, e.g., is more at the second time point than at the earlier first time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on the signs of worsening. In some embodiments, the subject receives treatment between two or more tests, and thus the test results indicate a treatment effect.

[0512] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments of the cancer cell, and, as described above, the methylation state can be used to determine the cell as being selected from fallopian epithelium, ovarian epithelium, and endometrial epithelium.

[0513] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0514] C. Cardiovascular-pulmonary cells C1. Alveolar epithelium Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in alveolar epithelial cells compared to all other cell types in humans.

[0515] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from an alveolar epithelial cell is provided. In some embodiments, the method involves detecting the methylation status of a plurality (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) of CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 2828-2838, 2839-2899, 2900-2900, 2901-2903, 2904-2916, or 2917-2953, or is located within a human genome sequence selected from SEQ ID NOs: 2828-2838 or 2839-2899, or within 100 bp, 200 bp, 500 bp, or 1 kb thereof. In some embodiments, the method then identifies the target DNA fragment as being from an alveolar epithelial cell if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from an alveolar epithelial cell if 25%, 30%, 35%, 40%, 45%, or 50% or less of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from an alveolar epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from an alveolar epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from an alveolar epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0516] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 2954-2960 or 2961-2978, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 2954-2960. In some embodiments, the method then identifies the target DNA fragment as being from an alveolar epithelial cell if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as being from an alveolar epithelial cell if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies a target DNA fragment as being from an alveolar epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies a target DNA fragment as not being from an alveolar epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as not being from an alveolar epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0517] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 2828-2838, 2839-2899, 2900-2900, 2901-2903, 2904-2916, 2917-2953, 2954-2960, or 2961-2978.

[0518] Example 2 of the present disclosure discloses a set of methylation markers that can distinguish different lung cell types, such as alveolar cells or bronchial cells. Exemplary markers are provided in Table 3. Seventeen genomic loci were uniquely unmethylated or hypermethylated in lung epithelial cells, including three loci that specifically identify bronchial cells, 12 loci that specifically identify alveolar cells, and two loci that can identify both. Using reference chromosomal locations as a reference, two loci that identify both bronchial cells and alveolar cells are chromosome 14: 55765534 (hg19; reference gene: FBXO34) and chromosome 3: 181441571 (reference gene: SOX2OT). The 12 loci that specifically identify 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), and chromosome 16: 1652552 (reference gene: :IFT140), chromosome 14:91691190 (reference gene:C14orf159), chromosome 16:667157 (reference gene:RAB40C), chromosome 11:66116455 (reference gene:B3GNT1), chromosome 4:57522145 (reference gene:HOPX), chromosome 16:84271391 (reference gene:KCNG4), and chromosome 1:1986275 (reference gene:PRKCZ). Three loci that specifically identify bronchial cells are chromosome 7:4802132 (reference gene:FOXK1), chromosome 2:239970075 (reference gene:HDAC4), and chromosome 1:164761834 (reference gene:PBX1).

[0519] For example, as shown in Figure 9, the genomic marker sequence in the Rab40C gene was unmethylated only in alveolar epithelium, but not in bronchial cells. As demonstrated in Figure 13, when the methylation status of one or more of these markers was used, lung cell types could be easily distinguished. When the top three markers were used, performance was close to that when all 17 markers were used, highlighting the robustness of this technique.

[0520] Thus, in one embodiment, there is provided a method for identifying that a biological sample contains DNA from a lung cell, the method comprising detecting a methylation state of each of at least four CpG sites of a target DNA fragment in the biological sample, and determining whether the methylation state corresponds to a reference human alveolar or bronchial cell, and whether the target DNA fragment corresponds to human chromosomes 14: 55765534, 3: 181441571, 1: 41486102, 2: 236672684, 17: 79952367, 16: 181441571, 1: 41486102, 2: 236672684, 16: 181441571 ... and identifying the target DNA fragment as being from a human alveolar cell or bronchial cell if the target DNA fragment is within 1 kb of a genomic locus selected from the group selected from: chromosome 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.

[0521] As used herein, in some embodiments, methylation status refers to the percentage of CpG sites that are methylated within a genomic sequence. In some embodiments, methylation status simply refers to hypermethylation (M, at least 60% CpG methylation) or hypomethylation (U, 40% or less CpG methylation).

[0522] For example, in one embodiment, the target DNA fragment is identified as being from a human alveolar cell if it is unmethylated and located near the genomic locus 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. In one embodiment, the target DNA fragment is identified as being from a human alveolar cell if it is methylated and located near the genomic locus Chromosome 4:57522145.

[0523] In one embodiment, the target DNA fragment is identified as being from a human lung bronchial cell if the target DNA fragment is unmethylated and located near the genomic locus Chromosome 7:4802132, Chromosome 2:239970075, or Chromosome 1:164761834.

[0524] In one embodiment, the target DNA fragment is identified as being from a human alveolar or bronchial cell if the target DNA fragment is unmethylated and located near the genomic locus chromosome 14:55765534 or chromosome 1:41486102, or if the target DNA fragment is methylated and located near the genomic locus chromosome 3:181441571.

[0525] In some embodiments, the DNA fragment containing the CpG site used in the measurement is within 1000 bp from a reference genomic location, for example, chromosome 14:55765534. In some embodiments, the DNA fragment containing the CpG site used in the measurement is within 900, 800, 700, 600, 500, 400, 300, 250, 200, or 150 bp from the reference genomic location.

[0526] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, if cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva, or cerebrospinal fluid) is identified as being from alveolar epithelial cells of the subject, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is alveolar epithelial damage, inflammation, or cancer.

[0527] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., alveolar epithelial cells, decreases, e.g., is less at the second time point than at the earlier first time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., alveolar epithelial cells, increases, e.g., is more at the second time point than at the earlier first time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of worsening. In some embodiments, the subject receives treatment between two or more tests, and thus the test results indicate a treatment effect.

[0528] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and the methylation state can be used to determine the cell as an alveolar epithelial cell, as described above.

[0529] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0530] C2. Pulmonary bronchial epithelium Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in lung bronchial epithelial cells compared to all other cell types in humans.

[0531] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from a lung bronchial epithelial cell is provided. In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 2979-3001, 3002-3087, 3088-3090, 3091-3092, or 3093-3104, or is located within a human genome sequence selected from SEQ ID NOs: 2979-3001 or 3002-3087, or within 100 bp, 200 bp, 500 bp, or 1 kb thereof. In some embodiments, the method then identifies the target DNA fragment as being from a lung bronchial epithelial cell if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a lung bronchial epithelial cell if 25%, 30%, 35%, 40%, 45%, or 50% or less of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a lung bronchial epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a lung bronchial epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from a lung bronchial epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0532] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 3105-3109 or 3110-3129, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 3105-3109. In some embodiments, the method then identifies the target DNA fragment as being from a lung bronchial epithelial cell if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as being from a lung bronchial epithelial cell if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies a target DNA fragment as being from a lung bronchial epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies a target DNA fragment as not being from a lung bronchial epithelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from a lung bronchial epithelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0533] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 2979-3001, 3002-3087, 3088-3090, 3091-3092, 3093-3104, 3105-3109, or 3110-3129.

[0534] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, if 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 pulmonary bronchial epithelial cells, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is pulmonary bronchial epithelial damage, inflammation, or cancer.

[0535] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., lung bronchial epithelial cells, decreases, e.g., is less at the second time point than at the earlier first time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., lung bronchial epithelial cells, increases, e.g., is more at the second time point than at the earlier first time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of worsening. In some embodiments, the subject receives treatment between two or more tests, and thus the test results indicate a treatment effect.

[0536] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and the methylation state can be used to identify the cell as a pulmonary bronchial epithelial cell, as described above.

[0537] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0538] C3. Cardiac cardiomyocytes Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in cardiac cardiomyocytes compared to all other cell types in humans.

[0539] 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 a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 3130-3147, 3148-3223, 3224-3230, or 3231-3254, or is located within a human genome sequence selected from SEQ ID NOs: 3130-3147 or 3148-3223, or within 100 bp, 200 bp, 500 bp, or 1 kb thereof. In some embodiments, the method then identifies the target DNA fragment as being from cardiac cardiomyocytes if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from cardiac cardiomyocytes if 25%, 30%, 35%, 40%, 45%, or 50% or less of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from cardiac cardiomyocytes if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from cardiac cardiomyocytes if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from cardiac cardiomyocytes if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0540] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites are located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genomic sequence selected from SEQ ID NOs: 3255-3266, 3267-3267, or 3268-3279, or selected from SEQ ID NOs: 3255-3266. In some embodiments, the method then identifies the target DNA fragment as being from a cardiac cardiomyocyte if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as being from a cardiac cardiomyocyte if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies a target DNA fragment as being from a cardiac cardiomyocyte if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies a target DNA fragment as not being from a cardiac cardiomyocyte if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as not being from a cardiac cardiomyocyte if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0541] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 3130-3147, 3148-3223, 3224-3230, 3231-3254, 3255-3266, 3267-3267, or 3268-3279.

[0542] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, if cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva, or cerebrospinal fluid) is identified as being from cardiac cardiomyocytes of the subject, the method indicates that the subject has aberrant cell death and / or a cell-related disease. In some embodiments, the disease or condition is cardiac cardiomyocyte injury, inflammation, or cancer.

[0543] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., cardiac cardiomyocytes, decreases, e.g., is less at the second time point of measurement than at the earlier first time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., cardiac cardiomyocytes, increases, e.g., is more at the second time point of measurement than at the earlier first time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of worsening. In some embodiments, the subject receives treatment between two or more tests, and thus the test results indicate a treatment effect.

[0544] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and the methylation state can be used to determine the cell as a cardiac cardiomyocyte, as described above.

[0545] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0546] C4. Cardiac fibroblasts Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in cardiac fibroblasts compared to all other cell types in humans.

[0547] According to one embodiment of the present disclosure, a method for identifying a biological sample as containing DNA from cardiac fibroblasts is provided. In some embodiments, the method involves detecting the methylation status of a plurality (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) of CpG sites of a target DNA fragment in the biological sample, wherein at least one (or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 33280-3300, 3301-3394, 3395-3396, 3397-3400, or 3401-3407, or selected from SEQ ID NOs: 3280-3300 or 3301-3394. In some embodiments, the method then identifies the target DNA fragment as being from cardiac fibroblasts if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from cardiac fibroblasts if 25%, 30%, 35%, 40%, 45%, or 50% or less of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from cardiac fibroblasts if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from cardiac fibroblasts if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from a cardiac fibroblast if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0548] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites are located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genomic sequence selected from SEQ ID NOs: 3408-3414, 3415-3416, or 3417-3432, or selected from SEQ ID NOs: 3408-3414. In some embodiments, the method then identifies the target DNA fragment as being from a cardiac fibroblast if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as being from a cardiac fibroblast if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies a target DNA fragment as being from a cardiac fibroblast if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies a target DNA fragment as not being from a cardiac fibroblast if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as not being from a cardiac fibroblast if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0549] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 3280-3300, 3301-3394, 3395-3396, 3397-3400, 3401-3407, 3408-3414, 3415-3416, or 3417-3432.

[0550] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, if cell-free DNA in a biological sample (e.g., blood, plasma, serum, semen, milk, urine, saliva, or cerebrospinal fluid) is identified as being from cardiac fibroblasts in the subject, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is cardiac fibroblast injury, inflammation, or cancer.

[0551] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., cardiac fibroblasts, decreases, e.g., is less at the second time point of measurement than at the earlier first time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., cardiac fibroblasts, increases, e.g., is more at the second time point of measurement than at the earlier first time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of worsening. In some embodiments, the subject receives treatment between two or more tests, and thus the test results indicate a treatment effect.

[0552] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and the methylation state can be used to determine the cell as a cardiac fibroblast, as described above.

[0553] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0554] C5. Vascular endothelial cells Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in vascular endothelial cells compared to all other cell types in humans.

[0555] According to one embodiment of the present disclosure, a method for identifying that a biological sample contains DNA from a vascular endothelial cell is provided. In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 3433-3456, 3457-3547, 3548-3550, 3551-3551, or 3552-3559, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 3433-3456 or 3457-3547. In some embodiments, the method then identifies the target DNA fragment as being from a vascular endothelial cell if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a vascular endothelial cell if 25%, 30%, 35%, 40%, 45%, or 50% or less of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a vascular endothelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a vascular endothelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from a vascular endothelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0556] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites are located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genome sequence selected from SEQ ID NOs: 3560-3579, 3580-3580, or 3581-3584, or selected from SEQ ID NOs: 3560-3579. In some embodiments, the method then identifies the target DNA fragment as being from a vascular endothelial cell if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as being from a vascular endothelial cell if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies a target DNA fragment as being from a vascular endothelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies a target DNA fragment as not being from a vascular endothelial cell if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies a target DNA fragment as not being from a vascular endothelial cell if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0557] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 3433-3456, 3457-3547, 3548-3550, 3551-3551, 3552-3559, 3560-3579, 3580-3580, or 3581-3584.

[0558] The cell type identification method can be used to detect diseases or conditions associated with the cell type. In one embodiment, if 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 vascular endothelial cells, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is vascular endothelial cell damage, inflammation, or cancer.

[0559] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., vascular endothelial cells, decreases, e.g., is less at the second time point than at the earlier first time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., vascular endothelial cells, increases, e.g., is more at the second time point than at the earlier first time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of worsening. In some embodiments, the subject receives treatment between two or more tests, and thus the test results indicate a treatment effect.

[0560] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and, as described above, the methylation state can be used to determine the cell as a vascular endothelial cell.

[0561] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0562] Group 8 - Cardiac cardiomyocytes and cardiac fibroblasts Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in cell populations, namely cardiac cardiomyocytes and cardiac fibroblasts, compared to all other cell types in humans.

[0563] According to one embodiment of the present disclosure, a method is provided for identifying a biological sample as containing DNA from cells selected from cardiac cardiomyocytes and cardiac fibroblasts. In some embodiments, the method involves detecting the methylation status of a plurality (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) of CpG sites in a target DNA fragment in the biological sample, wherein at least one (or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 6940-6959, 6960-7045, 7046-7046, 7047-7049, 7050-7053, or 7054-7065, or is located within or within 100 bp, 200 bp, 500 bp, or 1 kb of a human genome sequence selected from SEQ ID NOs: 6940-6959 or 6960-7045. In some embodiments, the method then identifies the target DNA fragment as being from a cell selected from cardiac cardiomyocytes and cardiac fibroblasts if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a cell selected from cardiac cardiomyocytes and cardiac fibroblasts if 25%, 30%, 35%, 40%, 45%, or 50% or less of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a cell selected from cardiac cardiomyocytes and cardiac fibroblasts if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not from a cell selected from cardiac cardiomyocytes and cardiac fibroblasts if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not from a cell selected from cardiac cardiomyocytes and cardiac fibroblasts if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0564] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 7066-7082 or 7083-7090, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genomic sequence selected from SEQ ID NOs: 7066-7082. In some embodiments, the method then identifies the target DNA fragment as being from a cell selected from a cardiac cardiomyocyte and a cardiac fibroblast if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a cell selected from cardiac cardiomyocytes and cardiac fibroblasts if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a cell selected from cardiac cardiomyocytes and cardiac fibroblasts if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a cell selected from cardiac cardiomyocytes and cardiac fibroblasts if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not from a cell selected from cardiac cardiomyocytes and cardiac fibroblasts if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0565] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 6940-6959, 6960-7045, 7046-7046, 7047-7049, 7050-7053, 7054-7065, 7066-7082, or 7083-7090.

[0566] The cell type identification method can be used to detect diseases or conditions associated with cell types. In one embodiment, if 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 cardiac cardiomyocytes and cardiac fibroblasts in the subject, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is injury, inflammation, or cancer of cells selected from cardiac cardiomyocytes and cardiac fibroblasts.

[0567] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., cells selected from cardiac cardiomyocytes and cardiac fibroblasts, decreases, e.g., is less at the second time point of measurement than at the earlier first time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., cells selected from cardiac cardiomyocytes and cardiac fibroblasts, increases, e.g., is more at the second time point of measurement than at the earlier first time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of worsening. In some embodiments, the subject receives treatment between two or more tests, and thus the test results indicate a treatment effect.

[0568] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and, as described above, the methylation state can be used to determine the cell as a cell selected from a cardiac cardiomyocyte and a cardiac fibroblast.

[0569] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0570] Group 9 - Alveolar and bronchial epithelium Also, as provided in Table A, several genomic locations are uniformly hypomethylated or hypermethylated in cell populations, namely, alveolar epithelium and pulmonary bronchial epithelium, compared to all other cell types in humans.

[0571] According to one embodiment of the present disclosure, a method is provided for identifying that a biological sample contains DNA from cells selected from alveolar epithelium and bronchial epithelium. In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NOs: 6227-6243, 6244-6326, 6327-6327, 6328-6329, 6330-6336, or 6337-6352, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genomic sequence selected from SEQ ID NOs: 6227-6243 or 6244-6326. In some embodiments, the method then identifies the target DNA fragment as being from a cell selected from alveolar epithelium and bronchial epithelium if 40% or less of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a cell selected from alveolar epithelium and bronchial epithelium if 25%, 30%, 35%, 40%, 45%, or 50% or less of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a cell selected from alveolar epithelium and bronchial epithelium if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not from a cell selected from alveolar epithelium and bronchial epithelium if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not from a cell selected from alveolar epithelium and bronchial epithelium if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated.

[0572] In some embodiments, the method involves detecting the methylation status of a plurality (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 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the CpG sites is selected from SEQ ID NO: 6353 or 6354-6365, or is located within, or within 100 bp, 200 bp, 500 bp, or 1 kb of, a human genomic sequence selected from SEQ ID NO: 6353. In some embodiments, the method then identifies the target DNA fragment as being from a cell selected from alveolar epithelium and pulmonary bronchial epithelium if 50% or more of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as being from a cell selected from alveolar epithelium and bronchial epithelium if at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are methylated. Similarly, in some embodiments, the method identifies the target DNA fragment as being from a cell selected from alveolar epithelium and bronchial epithelium if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are unmethylated. In some embodiments, the method identifies the target DNA fragment as not being from a cell selected from alveolar epithelium and bronchial epithelium if no more than 25%, 30%, 35%, 40%, 45%, or 50% of the CpG sites are methylated. In some embodiments, the method identifies the target DNA fragment as not being from a cell selected from alveolar epithelium and pulmonary bronchial epithelium if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the CpG sites are unmethylated.

[0573] In some embodiments, the methylation status of one or more other DNA fragments is further used in cell typing. In some embodiments, the one or more additional (different from the first) DNA fragments are represented by the genomic sequence of SEQ ID NOs: 6227-6243, 6244-6326, 6327-6327, 6328-6329, 6330-6336, 6337-6352, 6353, or 6354-6365.

[0574] The cell type identification method can be used to detect diseases or conditions associated with cell types. In one embodiment, if 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 subject's alveolar epithelium and bronchial epithelium, the method indicates that the subject has a disease associated with abnormal cell death and / or cells. In some embodiments, the disease or condition is injury, inflammation, or cancer of cells selected from the alveolar epithelium and bronchial epithelium.

[0575] In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., cells selected from alveolar epithelium and bronchial epithelium, is decreased, e.g., less at the second time point than at the earlier first time point of measurement, it indicates that the subject has recovered from the disease or condition. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of recovery. In some embodiments, if the amount of cell-free DNA identified as being from a particular cell type(s), e.g., cells selected from alveolar epithelium and bronchial epithelium, is increased, e.g., more at the second time point than at the earlier first time point of measurement, it indicates that the disease or condition has worsened. In some versions, the method includes making a diagnosis and / or treating the disease or condition appropriately based on signs of worsening. In some embodiments, the subject receives treatment between two or more tests, and thus the test results indicate a treatment effect.

[0576] In one embodiment, methods are also provided for determining the cell type of a diseased cell, e.g., a cancer cell, the primary origin of a cell of a disease, e.g., cancer, or the signal or origin of a cell of a disease, e.g., cancer. In some embodiments, the cancer cell is of unknown origin. In some embodiments, the method includes detecting the methylation state of one or more DNA fragments in the cancer cell, and, as described above, the methylation state can be used to determine that the cell is a cell selected from alveolar epithelium and pulmonary bronchial epithelium.

[0577] In some cases, cell-free DNA fragments are released from cancer cells. The technique may include determining the cell type of the cancer cells. In some embodiments, genetic variations may also be present in the DNA fragments, and thus detecting the cell type may help associate the genetic variation with cancer. In some embodiments, the genetic variation comprises a mutation. In some embodiments, the genetic variation comprises a deletion or 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 the generation of a premature stop codon. Once the primary origin of the cancer is identified, the subject may be treated with a regimen appropriate for the cancer type.

[0578] D. blood cells D1.Blood B cells Also, as provided in Table A, several genomic locations are...

Claims

1. 1. A method for identifying that a biological sample contains DNA from a cell type, comprising: detecting the methylation status of each of at least four CpG sites of a target DNA fragment in the biological sample; The target DNA fragment (1) identifying the cell as being from a human oral, laryngeal, or esophageal epithelial cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 1-90, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 126-133; (2) identifying the cell as being from a human gastric epithelial cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 151-330, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 379-401; (3) identifying the cell as being from a human small intestine epithelial cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 429 to 527, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 555 to 564; (4) identifying the cell as being from a human colon epithelial cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 580-657, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 705-715; (5) identifying the cell as being from a human colon fibroblast if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 730-732, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 733-739; (6) Identifying the cell as being from a human gallbladder epithelial cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 742-829, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 868-875; (7) Identifying the sample as being from a human liver hepatocyte if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 877-980, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 1003-1018; (8) Identifying the cell as being from a human pancreatic acinar cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 1028-1112, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 1156-1161; (9) identifying the cell as being from a human pancreatic alpha cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 1181-1282, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 1307-1315; (10) Identifying as being from a human pancreatic beta cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 1332-1440, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 1461-1471; (11) Identifying as from a human pancreatic delta cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 1486-1594, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 1614-1624; or (12) Identifying the cells as being from a human pancreatic duct cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 1639-1742, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 1768-1779.

2. detecting the methylation status of each of at least four CpG sites of a second target DNA fragment in the biological sample; and (1') identifying the cell as being from a human oral, laryngeal, or esophageal epithelial cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 1 to 125, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 126 to 150; (2') identifying the cell as being from a human gastric epithelial cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 151 to 378, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 379 to 428; (3') identifying the cell as being from a human small intestine epithelial cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 429 to 554, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 555 to 579; (4') identifying the cell as being from a human colon epithelial cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 580-704, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 705-729; (5') identifying the cell as being from a human colon fibroblast if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 730-732, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 733-741; (6') identifying the cell as being from a human gallbladder epithelial cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 742 to 867, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 868 to 876; (7') Identifying the sample as being from a human liver hepatocyte if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 877-1002, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 1003-1027; (8') identifying the cell as being from a human pancreatic acinar cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 1028-1155, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 1156-1180; (9') identifying the cell as being from a human pancreatic alpha cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 1181-1306, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 1307-1331; (10') identifying the cell as being from a human pancreatic beta cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 1332-1460, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 1461-1485; (11') identifying the cell as being from a human pancreatic delta cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 1486-1613, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 1614-1638; or (12') If 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 1639 to 1767, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 1768 to 1792, the method of claim 1 further comprising identifying the cell as being from a human pancreatic duct cell.

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

4. 4. The method of claim 3, wherein the target DNA fragments are cell-free DNA fragments.

5. 5. The method of claim 4, wherein identifying the cell-free DNA fragments as being from a cell type comprises detecting aberrant cell death of the cell type or a disease associated with the cell type.

6. 5. The method of claim 4, further comprising identifying the human subject as having or likely to have injury, inflammation, or cancer in the corresponding cell type.

7. 6. The method of claim 5, further comprising identifying the human subject as having or likely to have a pancreatic disease or condition if the amount of cell-free DNA fragments identified as being from a pancreatic cell type is greater than a reference cutoff value.

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

9. 1. A method for identifying that a biological sample contains DNA from a cell type, comprising: detecting the methylation status of each of at least four CpG sites of a target DNA fragment in the biological sample; The target DNA fragment (1) identifying the cell as being from a human endometrial epithelial cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 1793-1864, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 1893-1905; (2) identifying the cell as being from a human fallopian epithelial cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 1918-2022, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 2043-2061; (3) identifying the cell as being from a human kidney epithelial cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 2068-2141, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 2195-2209; (4) identifying the cell as being from a human bladder epithelial cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 2220-2298, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 2346-2350; (5) Identifying as being from a human prostate epithelial cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 2371-2476, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 2496-2500; (6) identifying the cell as being from a human breast basal epithelial cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 2521-2616, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 2652-2659; or (7) Identifying the cells as being from human breast luminal epithelial cells if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 2677-2748, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 2803-2815.

10. 10. The method of claim 9, wherein the biological sample comprises blood, plasma, serum, semen, milk, urine, saliva, or cerebrospinal fluid obtained from a human subject.

11. 11. The method of claim 10, wherein the target DNA fragments are cell-free DNA fragments.

12. 12. The method of claim 11, wherein identifying the cell-free DNA fragments as being from a cell type indicates aberrant cell death of the cell type or a disease associated with the cell type.

13. 12. The method of claim 11, further comprising identifying the human subject as having or likely to have injury, inflammation, or cancer in corresponding urogenital cells.

14. 1. A method for identifying that a biological sample contains DNA from a cell type, comprising: detecting the methylation status of each of at least four CpG sites of a target DNA fragment in the biological sample; The target DNA fragment (1) identifying the cell as being from a human alveolar epithelial cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 2828-2899, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 2954-2960; (2) identifying the cell as being from a human lung bronchial epithelial cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 2979-3087, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 3105-3109; (3) identifying the cell as being from a human cardiac cardiomyocyte if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 3130-3223, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 3255-3266; (4) identifying the cell as being from a human cardiac fibroblast if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 3280-3394, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 3408-3414; or (5) identifying the cell as being from a human vascular endothelial cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 3433-3547, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 3560-3579.

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

16. 16. The method of claim 15, wherein the target DNA fragments are cell-free DNA fragments.

17. 17. The method of claim 16, wherein identifying the cell-free DNA fragments as being from a cell type indicates abnormal cell death of the cell type or disease in a corresponding cardiovascular / pulmonary cell.

18. 1. A method for identifying that a biological sample contains DNA from a cell type, comprising: detecting the methylation status of each of at least four CpG sites of a target DNA fragment in the biological sample; The target DNA fragment (1) identifying the sample as being from a human B cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 3585-3701, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 3713-3733; (2) identifying the sample as being from a human granulocyte if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 3738-3849, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 3863-3884; (3) identifying the nucleic acid as being from a human monocyte or macrophage if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 3887-3997, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 4013-4036; (4) identifying the cells as being from human natural killer (NK) cells if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 4038-4146, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 4163-4184; (5) identifying the T cell as being from a human T cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 4188-4274, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 4313-4322; or (6) identifying the cell as being from a human erythroid precursor cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 4338-4449, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 4465-4470.

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

20. 20. The method of claim 19, wherein the target DNA fragments are cell-free DNA fragments.

21. 21. The method of claim 20, wherein identifying the cell-free DNA fragments 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. 21. The method of claim 20, further comprising identifying the human subject as having or likely to have an autoimmune disease, inflammation, infection, or cancer.

23. 1. A method for identifying that a biological sample contains DNA from a cell type, comprising: detecting the methylation status of each of at least four CpG sites of a target DNA fragment in the biological sample; The target DNA fragment (1) identifying the cells as being from human epidermal keratinocytes if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 4471 to 4573, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 4596 to 4598; (2) identifying the cell as being from a human skin fibroblast if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 4619 to 4719, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 4742 to 4747; (3) identifying the cell as being from a human osteoblast if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 4767-4869, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 4892-4897; (4) identifying the cell as being from a human skeletal muscle cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 4917-5016, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 5041-5043; or (5) identifying the cell as being from a human smooth muscle cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 5065-5178, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 5192-5204.

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

25. 25. The method of claim 24, wherein the target DNA fragments are cell-free DNA fragments.

26. 26. The method of claim 25, wherein identifying the cell-free DNA fragments as being from a cell type indicates aberrant cell death of the cell type or a disease associated with the cell type.

27. 27. The method of claim 26, further comprising identifying the human subject as having or likely to have inflammation or cancer in corresponding dermal-skeletal-muscle cells.

28. 1. A method for identifying that a biological sample contains DNA from a cell type, comprising: detecting the methylation status of each of at least four CpG sites of a target DNA fragment in the biological sample; The target DNA fragment (1) identifying the cell as being from a human thyroid epithelial cell if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 5217-5284, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 5344-5358; (2) identifying the sample as being from a human adipocyte if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 5369-5445, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genome sequence selected from the group consisting of SEQ ID NOs: 5454-5463; (3) identifying the cell as being from a human neuron if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 5471-5556, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 5595-5613; or (4) identifying the cells as being from a human oligodendrocyte if 40% or less of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 5620-5721, or if 50% or more of the CpG sites are methylated and at least one of the CpG sites is located in a human genomic sequence selected from the group consisting of SEQ ID NOs: 5772-5782.

29. 29. The method of claim 28, wherein the biological sample comprises blood, plasma, serum, semen, milk, urine, saliva, or cerebrospinal fluid obtained from a human subject.

30. 30. The method of claim 29, wherein the target DNA fragments are cell-free DNA fragments.

31. 31. The method of claim 30, wherein identifying the cell-free DNA fragments as being from a cell type indicates aberrant cell death of the cell type or a disease associated with the cell type.

32. 31. The method of claim 30, further comprising identifying the human subject as having or likely to have multiple sclerosis (MS) if the biological sample contains target DNA fragments identified as being from oligodendrocytes.

33. 31. The method of claim 30, further comprising identifying the human subject as having or likely to have a neurodegenerative disorder if the biological sample contains target DNA fragments identified as being from neuronal cells.

34. 1. A method for identifying that a biological sample contains DNA from a lung cell, comprising detecting a methylation state of each of at least four CpG sites of a target DNA fragment in the biological sample, wherein the methylation states correspond to reference human alveolar or bronchial cells, and the target DNA fragments correspond to the following sequences according to human genome assembly version hg19: human chromosome 14: 55765534, chromosome 3: 181441571, chromosome 1: 41486102, chromosome 2: 236672684, chromosome 17: 79952367, chromosome 16: 678127, and identifying the target DNA fragment as being from a human alveolar cell or bronchial cell if the target DNA fragment is within 1 kb of a genomic locus selected from the group selected from: 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. (a) the target DNA fragment is identified as being from a human alveolar cell if 40% or less of the CpG sites are methylated and the target DNA fragment is within 1 kb 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, or if at least 60% of the CpG sites are methylated and the target DNA fragment is within 1 kb of Chromosome 4:57522145; (b) the target DNA fragment is identified as being from a human lung bronchial cell if 40% or less of the CpG sites are methylated and the target DNA fragment is within 1 kb of chromosome 7: 4802132, chromosome 2: 239970075, or chromosome 1: 164761834; or (c) the target DNA fragment is identified as being from a human alveolar or bronchial cell if 40% or less of the CpG sites are methylated and the target DNA fragment is within 1 kb of chromosome 14:55765534 or chromosome 1:41486102, or if at least 60% of the CpG sites are methylated and the target DNA fragment is within 1 kb of chromosome 3:181441571.

36. 2. The method of claim 1, wherein the target DNA fragment has a length of 50 to 200 bp.

37. 2. The method of claim 1, wherein the methylation state is the conversion of cytosine to 5-methylcytosine (5-mC) or 5-hydroxymethylcytosine (5-hmC).

38. 38. The method of claim 37, wherein detecting the methylation status comprises bisulfite or enzymatic treatment of the DNA fragments, or digestion of the DNA fragments with a restriction enzyme sensitive to DNA methylation.

39. 39. The method of claim 38, wherein the enzymatic treatment comprises treatment with APOBEC-Seq.

40. 39. The method of claim 38, wherein detecting the methylation status further comprises determining the sequence of the DNA fragment.

41. 41. The method of claim 40, wherein the sequence is determined by deep sequencing.

42. 39. The method of claim 38, wherein detecting the methylation status further comprises detecting digested fragments.

43. 10. The method of claim 1, further comprising detecting a genetic mutation in the target DNA fragment, thereby determining that the cell into which the target DNA fragment is released contains the genetic mutation.

44. 6. The method of claim 5, further comprising administering to the patient an agent useful for treating the identified disease or condition.

45. 45. A method for identifying a cell type of a cancer cell, the method comprising identifying the cell type of the cancer cell using the method of any one of claims 1 to 44.

46. 46. ​​The method of claim 45, further comprising detecting a genetic mutation in the genomic DNA of the cancer cell.

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

48. 48. The method of claim 47, further comprising locating a tissue origin of the cancer cells based on the cell type.

49. 46. ​​The method of claim 45, further comprising treating cancer in the subject from which the cancer cells were obtained.

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