Panels and methods for identifying cell origin of cell free DNA

Abstract

Described herein is a method of identifying cell origin of cell free DNA molecules in a biological sample from a subject, the method comprising: determining a level of methylation for one or more target polynucleotide regions listed in Table 1 in the cell free DNA molecules; and identifying the cell origin of the cell free DNA molecules based on the methylation level of the one or more the one or more target polynucleotide regions. Also described is at least one primer or probe complementary to one or more of target sequence comprised in one or more regions listed in Table 1 and panels comprising a plurality of the at least one primers or probes.

Description

PANELS AND METHODS FOR IDENTIFYING CELL ORIGIN OF CELL FREE DNACROSS REFERENCE

[0001] This PCT application claims the benefit to U.S. Provisional Application No. 63 / 632,407, filed April 10, 2024, the content of which is incorporated herein by reference.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] This disclosure contains a Sequence Listing, which has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The accompanying Sequence Listing file named “062649-507001 WO_Sequence Listing ST26.xml” was created on April 1 , 2025.FIELD

[0003] The present disclosure relates to panels and methods for identifying cell origin of cell free DNA, in particular using methylation levels of cell free DNA.BACKGROUND

[0004] DNA methylation in humans predominantly occurs on cytosines in a CpG dinucleotide, where the cytosine on both strands is usually methylated or unmethylated by the addition of a methyl- or hydroxymethyl- group to form 5- methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC), respectively. The methylation patterns in a given genome are associated with the type and state of the cell. Consecutive CpGs within a specific distance from each other and with similar methylation rates can be grouped together into regions of CpG methylation. The methylation rates across all of the CpGs in these regions are typically averaged producing an average CpG methylation rate for each region. The most useful of these regions are those that are hypo- (mostly unmethylated) or hyper- (mostly methylated) methylated in a specific cell type or state, for example, lung AT2 cells or activated T- cells. These tissue specific hypo- and hyper- methylated regions can be used to deconvolute a sample of DNA consisting of a mixture of cell-types, for example, plasma cell-free DNA or whole tissue into the proportion of each of the cell types contributing to the mixture.

[0005] Cell-free DNA are small DNA fragments released from cells as they die. These DNA fragments are typically protected from further degradation by anucleosome or other DNA binding protein. The challenge with cell-free DNA is that the DNA fragment size is generally small (~168bp), which limits the number of CpGs that can be detected from a single fragment. This issue is even more challenging when using a targeted panel where the primers may have reduced sensitivity depending on the CpG’s location in the cell-free DNA molecule and the primer(s) location in the fragment.

[0006] In the context of organ transplant, current commercial tests quantify the proportion of donor derived cfDNA (ddcfDNA %). An increase in ddcfDNA can indicate graft damage. However, an increase in ddcfDNA cannot differentiate infection of a transplanted organ from rejection of a transplanted organ. There is a need for additional context to better differentiate rejection and infection. Existing commercial tests utilize cell-free DNA in the post-transplant period to predict donor organ injury by the identification of single nucleotide variants (SNVs) that are donor specific. This approach allows for the recognition of donor organ injury but less about how the organ is being injured.

[0007] Existing commercial tests examining cell-free DNA (cfDNA) rely on panels of pre-selected single nucleotide variations (SNV) chosen to likely occur between random donor / recipient pairings. Following transplantation, a % or absolute amount of donor-derived cfDNA (ddcfDNA) is calculated, with the presumption that an increased amount of ddcfDNA reflects injury to the graft. While this may indicate graft injury, a limitation is that it does not indicate the etiology of the injury, and thus clinicians will not know how to treat it (i.e., is the graft injury from T-cell rejection, antibody-mediated rejection, infection, or some other cause). Another limitation is technical; because the donor and recipient are not genotyped, whether a set of SNVs is donor-origin or recipient-origin is inferred. Moreover, whether a SNV is homozygous or heterozygous is unknown. Consequently, when % ddcfDNA is low, the accuracy can be high; however, when the %ddcfDNA begins to surpass 15-20%, the accuracy will fall and panel-based tests are inaccurate above 25% or 30%. Accordingly, there is a need for alternate modalities.SUMMARY

[0008] To enable accurate cell-of-origin profiling from cell-free DNA samples, the inventors developed an assay to find tissue specific hypo- and hyper- methylatedregions that are constrained to small regions of hypo- and hyper- CpGs methylation that maximizes their detection using a targeted panel for cell-free DNA samples.

[0009] As described herein, the methylation pattern of cfDNA target fragments is used in the methods described herein to predict what organ the cfDNA fragment comes from. This method can for example recognize donor organ injury (e.g., cfDNA from the transplanted organ type) and inform about cause of donor organ injury based on cfDNA cell origin patterns (e.g., high T-cell origin indicates rejection).

[0010] In particular, described herein is a targeted panel of methylation sites predictive of cell origin. Patient plasma, for example, can be used with this panel and the cell origin pattern determined.

[0011] Accordingly, an aspect of the disclosure includes a method of identifying cell origin of cell free DNA molecules in a biological sample, the method comprising: determining a level of methylation for one or more target polynucleotides listed in Table 1 in the cell free DNA molecules; and identifying the cell origin of the cell free DNA molecules based on the methylation level of the one or more the one or more target polynucleotide.

[0012] Another aspect of the disclosure includes a method of diagnosing organ injury, the method comprising: a) providing a biological sample; b) isolating cell free DNA molecules from the biological sample; c) determining a level of methylation for one or more target polynucleotide regions listed in Table 1 in the cell free DNA molecules; d) identifying cell origin of the cell free DNA molecules based on the methylation level of the one or more the one or more target polynucleotide regions, thereby diagnosing organ injury.

[0013] In some embodiments, the organ injury comprises a rejection of a transplant organ. In some embodiments, the organ injury comprises inflammation. In some embodiments, the organ injury comprises organ trauma.

[0014] Another aspect of the disclosure includes a method of predicting a transplant outcome, comprising: a) providing a biological sample; b) isolating cell free DNA molecules from the biological sample; c) determining a level of methylation for one or more target polynucleotide regions listed in Table 1 in the cell free DNA molecules; d) identifying the cell origin of the cell free DNA molecules based on themethylation level of the one or more the one or more target polynucleotide regions, thereby predicting the transplant outcome.

[0015] In some embodiments, the transplant outcome comprises rejection of a transplant organ. In some embodiments, the transplant outcome comprises transplant of a transplant organ.

[0016] Another aspect of the disclosure includes one or more primer or probe complementary to one or more target sequence, wherein the one or more target sequence is comprised in one or more regions listed in Table 1 .

[0017] Another aspect of the disclosure includes a composition comprising the one or more primer or probe described herein.

[0018] Another aspect of the disclosure includes a panel comprising a plurality of the one or more primers or probes described herein or any composition described herein.

[0019] Another aspect of the disclosure includes a kit comprising any described herein or a plurality of the one or more primer or probe described herein or any composition described herein.

[0020] The preceding section is provided by way of example only and is not intended to be limiting on the scope of the present disclosure and appended claims. Additional objects and advantages associated with the compositions and methods of the present disclosure will be appreciated by one of ordinary skill in the art in light of the instant claims, description, and examples. For example, the various aspects and embodiments of the disclosure may be utilized in numerous combinations, all of which are expressly contemplated by the present description. These additional advantages objects and embodiments are expressly included within the scope of the present disclosure. The publications and other materials used herein to illuminate the background of the disclosure, and in particular cases, to provide additional details respecting the practice, are incorporated by reference, and for convenience are listed in the appended reference section.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Further objects, features and advantages of the disclosure will become apparent from the following detailed description taken in conjunction with the accompanying figures showing illustrative embodiments of the disclosure, in which:

[0022] Fig. 1 depicts heatmap of methylation of a lung region and a graph showing the level of methylation at various positions. (Top) An exemplary methylation pattern of a specific part of the genome. (Bottom) An exemplary CpG hypomethylated region derived from alveolar cells from the lung.

[0023] Fig . 2 depicts a heatmap generated from a publicly available tissue atlas, showing hypomethylated specific regions from multiple cell-types. Each row is a sample from a specific cell-type as indicated by the y-axis labels. Each column is a hypomethylated region specific to a single cell-type

[0024] Fig. 3 depicts a heatmap showing % tissue contribution of different cells with different origins within different cell types.

[0025] Fig. 4 depicts graphs depicting presence and size of cell free DNA in 4h perfusate sample and in post-transplant plasma sample. Mono-, Di-, and Tri- refer to mononucleosomes, dinucleosomes, and trinucleosomes.

[0026] Fig. 5A depicts a heatmap showing percent tissue contribution of different cell types in an EVLP rejected sample and an EVLP transplanted sample.

[0027] Fig. 5B depicts a graph showing percent of tissue contribution of cells with a cell origin from monocytes and granulocytes in rejected samples and transplanted samples.

[0028] Fig. 6 depicts schematics of exemplary reads from a panel described herein.

[0029] Fig. 7 depicts a heatmap showing tissue contribution % origin for 3 samples taken 24 post-transplant, 3 samples taken from perfusate, 1 sample using lung tissue, and 1 sample with esophageal cancer determined using the methods described herein.

[0030] Fig. 8 depicts graphs showing results of panel validation with matched EM-seq sequencing.

[0031] Fig. 9 depicts graphs showing results of CpG coverage in different samples.

[0032] Fig. 10 depicts a heatmap showing % tissue contribution of cells from different origins in a plasma sample take 24 hours post-transplant, and a sample of healthy plasma.DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS

[0033] The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the disclosure. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated by reference in their entirety.

[0034] The following non-limiting examples are illustrative of the present application:I. Definitions

[0035] As used herein, the following terms may have meanings ascribed to them below, unless specified otherwise. However, it should be understood that other meanings that are known or understood by those having ordinary skill in the art are also possible, and within the scope of the present disclosure. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0036] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the description. Ranges from any lower limit to any upper limit are contemplated. The upper and lower limits of these smaller ranges which may independently be included in the smaller ranges is also encompassed within the description, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the description.

[0037] The term “about” as used herein may be used to take into account experimental error and variations that would be expected by a person having ordinary skill in the art. For example, “about” may mean plus or minus 10%, or plus or minus 5%, of the indicated value to which reference is being made.

[0038] As used herein the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise.

[0039] The phrase "and / or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to those elements specifically identified.

[0040] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of" or, when used in the claims, "consisting of" will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of."

[0041] As used herein, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of” and "consisting essentially of” shall be closed or semi-closed transitional phrases, respectively.

[0042] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listedwithin the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.

[0043] As used herein, a “biological sample” can refer to any cell free sample obtained from a biological source a subject or flushed through a biological source from a subject (e.g. donor organ) from which cell free nucleic acids e.g., cell free DNA, can be obtained. The biological sample can be a sample obtained directly from a biological source (e.g., plasma, serum, urine, etc.) or a sample that is processed (e.g., tissue perfusate flushed through an organ).

[0044] The term “nucleic acid” as used herein refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil as well as inosine. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine. The nucleic acid can be either double stranded or single stranded, and represents the sense or antisense strand. Further, the term “nucleic acid” includes the complementary nucleic acid sequences as well as codon optimized or synonymous codon equivalents. The nucleic acids can for example or comprise one or more locked nucleic acid (LNA) moieties. The term “isolated nucleic acid molecules” as used herein refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized. An isolated nucleic acid is also substantially free of sequences which naturally flank the nucleic acid (i.e. sequences located at the 5' and 3' ends of the nucleic acid) from which the nucleic acid is derived.

[0045] The term “detectable label” as used herein refers to moieties such as peptide sequences, fluorescent proteins that can be appended or introduced into a peptide, antibody or other compound described herein and which is capable ofproducing, either directly or indirectly, a detectable signal. For example, the label may be radio-opaque, or a radioisotope; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin. For example, the detectable label may be one useful for qPCR or microarray.

[0046] “Hybridization” as used herein may refer to the process in which two single-stranded polynucleotides bind non-covalently to form a stable double- stranded polynucleotide.

[0047] The term “methylation” in the context of DNA refers to the level, e.g. presence or absence or hypo or hyper, of 5-methylcytosine (“5-mC” or“5-mCyt”) and / or 5-hydroxymethylcytosine (“5-hmC” or “5-hmCyt”) collectively referred to as “5-mCyt” herein at one or a plurality of CpG dinucleotides within a DNA sequence or region. Methylation profiles of a DNA sequence include hypermethylation and hypomethylation. The term “hypomethylation” refers to the average methylation state corresponding to a decreased presence of 5-mCyt at one or a plurality of CpG dinucleotides within a DNA sequence of a test biological sample, relative to the amount of 5-mCyt found at corresponding CpG dinucleotides within a control biological sample and can for example refer to an average methylation rate of less than 0.50 e.g., 0.33.

[0048] As used herein, “ex vivo perfusion of organs” refers to techniques or procedures for maintaining organ viability within or outside a host e.g., ex-vivo lung perfusion. The methods described herein include pumping a perfusate or medical fluid through an organ and delivering one or more gene editing reagents. The organ can be subjected to normothermic perfusion, hypothermic perfusion, or perfusion at room temperature.

[0049] As referred to herein, the term “perfusate” refers to a fluid used in perfusion of organs or tissue. The medical solution can be, for example, Belzer's Gluconate-Albumin Solution, Steen solution, University of Wisconsin Solution, histidine-tryptophan-ketoglutarate solution, blood, Lifor, or AQIX-RS-I. In some embodiments, the solution can further comprise an oxygen carrier, including perfluorocarbon and hemoglobin-based oxygen carriers.

[0050] As used herein, the term “cell free DNA” or “cfDNA” refers to partially degraded or cleaved endogenous DNA of the body free from cells, resulting for example from apoptosis or necrosis of cells.

[0051] As used herein, the term “donor-derived cell-free DNA” or “ddcfDNA” refers to the cell-free (non- encapsulated) DNA derived from apoptosis or necrosis of allograft tissue, which circulates in the body fluids of patients which have received an organ transplant, after organ transplantation, but which is derived from tissue from the organ donor e.g., the organ that has been transplanted.II. Primers, Probes and Panels

[0052] cfDNA fragments retain information from their cell of origin. The inventors as reported here, deconvoluted a cfDNA methylation profile to determine the originating tissues / cells.

[0053] Another aspect of the disclosure includes an isolated polynucleotide comprising a sequence of any one of the regions listed in Table 1 or a portion thereof.

[0054] An aspect of the disclosure includes at least one primer or probe, optionally a plurality of primers or probes, complementary to one or more target sequence, wherein each of the one or more target sequence is comprised in a region listed in Table 1 . In some embodiments, the plurality of primers or probes comprise the nucleotide sequence of any one of SEQ ID NOs: 1 -2921 or at least 15 consecutive residues of any thereof. In some embodiments, the plurality of primers or probes comprise the nucleotide sequence of any one of SEQ ID NOs: 1 -2921 or at least 16 consecutive residues of any thereof. In some embodiments, the plurality of primers or probes comprise the nucleotide sequence of any one of SEQ ID NOs: 1 -2921 or at least 17 consecutive residues of any thereof. In some embodiments, the plurality of primers or probes comprise the nucleotide sequence of any one of SEQ ID NOs: 1 - 2921 or at least 18 consecutive residues of any thereof. In some embodiments, the plurality of primers or probes comprise the nucleotide sequence of any one of SEQ ID NOs: 1 -2921 or at least 19 consecutive residues of any thereof. In some embodiments, the plurality of primers or probes comprise the nucleotide sequence of any one of SEQ ID NOs: 1 -2921 or at least 20 consecutive residues of any thereof. In some embodiments, the regions listed in Table 1 are regions identified as being hypo- methylated in a particular cell type. In some embodiments, the one or more target sequence is a portion of the region identified as being hypo-methylated in a particular cell type (e.g., the regions listed in Table 1 ).

[0055] Each primer or probe can be complementary to the sense strand or complementary the antisense strand. Each primer can be a forward primer or a reverse primer. In some embodiments, a primer or probe of the plurality of the primers or probes is an isolated primer or probe. In some embodiments, the plurality of primers or probes are isolated primers or probes.

[0056] In some embodiments, each primer or probe comprises at least about 15 nucleotides. In some embodiments, each primer or probe comprises at least about 20 nucleotides. In some embodiments, each primer or probe comprises up to about 50 nucleotides. In some embodiments, each primer or probe comprises between about 15 nucleotides and about 50 nucleotides. In some embodiments, each primer or probe comprises between about 20 nucleotides and about 50 nucleotides. In some embodiments, each primer or probe comprises about 15 nucleotides, about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 35 nucleotides, about 40 nucleotides, about 45 nucleotides, and / or about 50 nucleotides. In some embodiments, each primer or probe comprises about 20 nucleotides.

[0057] Another aspect of the disclosure includes a panel comprising a plurality of primers or probes complementary to one or more target sequence, wherein the one or more target sequence is comprised in one or more regions listed in Table 1 . In some embodiments, the one or more target sequence is a plurality of target sequences. In some embodiments, the plurality of primers or probes are fixed to a substrate. In some embodiments, the substrate is a solid surface, optionally a chip, plate, beads, or slide.

[0058] In some embodiments, the at least one or plurality of primers or probes comprise the nucleotide sequence of any one of SEQ ID NOs: 1 -2921 or at least 15 consecutive residues of any thereof. In some embodiments, the at least one or plurality of primers or probes comprise the nucleotide sequence of any one of SEQ ID NOs: 1 - 2921 or at least 20 consecutive residues of any thereof. In some embodiments, the at least one or plurality of primers or probes are isolated primers or probes.

[0059] The at least one or plurality of primers or probes or the panel thereof can also be comprised in a composition. For example, in some embodiments, the composition is provided in a well. In some embodiments, the composition is provided in a plate. In some embodiments, the composition is lyophilized. In some embodiments, the composition is frozen.

[0060] The target sequence is for example at least 20 consecutive residues comprised in any of the regions listed in Table 1 . Primers complementary to these target sequences can be used to amplify the regions identified in Table 1 or a portion thereof comprising at least one CpG motif. For example, the at least one primer or a pair of primers can be used to amplify at least one of the regions listed in Table 1 or at least or a portion thereof comprising at least one CpG motif. In some embodiments, the at least one primer or pair of primers can be used to amplify a plurality of the regions listed in Table 1 or at least a portion of each thereof comprising at least one CpG motif. In some embodiments, the at least one primer or pair of primers can be used to amplify a plurality of the regions listed in Table 1 or at least a portion of each of the plurality of the nucleic acid sequences thereof comprising at least one CpG motif. In some embodiments, the at least one primer can be used to amplify at least 2 nucleotides of any one of the regions listed in Table 1 . For example, the at least one primer can be used to amplify at least one CpG motif of at least one of the regions listed in Table 1 . In some embodiments, the at least one primer can be used to amplify one CpG motif. In some embodiments, the at least one primer can be used to amplify a plurality of CpG motifs. In some embodiments, the at least one primer can be used to amplify between 1 to 70 CpG motifs. In some embodiments, the at least one primer can be used to amplify 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 CpG motifs. In some embodiments, the at least one primer can be used to amplify about 1 , about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65 or about 70 CpG motifs.

[0061] In some embodiments, the panel comprises a subset of the plurality of primers or probes comprising the nucleotide sequence of any one of SEQ ID NOs: 1 - 2921 , or least 20 consecutive residues of any thereof. In some embodiments, the panel is specific to one or more tissue or cell type (e.g., a panel for the kidney, lung, liver, and / or pancreas), wherein the primers or probes of the panel are complementary to one or more target sequence comprised in one or more regions listed Table 1 , which exhibit specific methylation patterns in one or more particular tissue or cell type (e.g., kidney, lung, liver, and / or pancreas).

[0062] In some embodiments, the plurality of primers comprises at least 10 primers, at least 11 primers, at least 12 primers, at least 13 primers, at least 14 primers, at least 15 primers, at least 16 primers, at least 17 primers, at least 18primers, at least 19 primers, or at least 20 primers. In some embodiments, the plurality of primers comprises at least 20 primers. In some embodiments the plurality of primers comprises up to 100 primers. In some embodiments the plurality of primers comprises between 10 to 100 primers. In some embodiments, the plurality of primers comprises at least 10 primers.

[0063] In some embodiments, the panel comprises at least one primer or probe per tissue type in the panel. In some embodiments, the panel comprises at least 2 primers or probes per tissue type in the panel. In some embodiments, the panel comprises at least 3 primers or probes per tissue type in the panel. In some embodiments, the panel comprises at least 4 primers or probes per tissue type in the panel. In some embodiments, the panel comprises at least 5 primers or probes per tissue type in the panel. In some embodiments, the panel comprises at least 6 primers or probes per tissue type in the panel. In some embodiments, the panel comprises at least 7 primers or probes per tissue type in the panel. In some embodiments, the panel comprises at least 8 primers or probes per tissue type in the panel. In some embodiments, the panel comprises at least 9 primers or probes per tissue type in the panel. In some embodiments, the panel comprises a plurality of primers or probes per tissue type in the panel. In some embodiments, the plurality of primers or probes comprises at least 10 primers or probes per tissue type in the panel. In some embodiments, the plurality of primers or probes comprises at least 10 primers or probes, at least 11 primers or probes, at least 12 primers or probes, at least 13 primers or probes, at least 14 primers or probes, at least 15 primers or probes, at least 16 primers or probes, at least 17 primers or probes, at least 18 primers or probes, at least 19 primers or probes, or at least 20 primers or probes per tissue type in the panel. In some embodiments, the plurality of primers comprises at least 20 primers per tissue type in the panel. In some embodiments the plurality of primers or probes comprises up to 100 primers or probes per tissue type in the panel. In some embodiments the plurality of primers or probes comprises between 10 to 100 primers or probes per tissue type in the panel. In some embodiments, the panel comprises primers or probes for one or more tissue type. In some embodiments, the panel comprises primers or probes for one tissue type. In some embodiments, the panel comprises primers or probes for a plurality of tissue types.

[0064] In some embodiments, each or a subset of the plurality of primers or probes can comprise a tag. In some embodiments, the tag comprises a common sequence, a unique molecular identifier, or an index tag.

[0065] In some embodiments, the at least one or plurality of primers or probes will hybridize with a complementary sequence under suitable conditions. Suitable hybridization buffers and conditions are well known to those of skill in the art and are described, for example, in Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainview, N.Y. (1989); and Ausubel et al, Short Protocols in Molecular Biology, 4th ed., John Wiley & Sons (1999).

[0066] In some embodiments, the at least one or plurality of primers, optionally a forward primer or a reverse primer, is complementary to at least 20 nucleotides or more of one or more target sequence described herein. In some embodiments, the at least one or plurality of primers, optionally a forward primer or a reverse primer, have a length of 45 nucleotides or less. In some embodiments, the at least one or plurality of primers, optionally a forward primer or a reverse primer, have a length of between 20 and 45 nucleotides.

[0067] In some embodiments, the at least one or plurality of primers or probes comprises at least about 20 nucleotides. In some embodiments, the at least one or plurality of primers or probes comprises up to about 50 nucleotides. In some embodiments, the at least one or plurality of primers or probes comprises between about 20 nucleotides and about 50 nucleotides. In some embodiments, the at least one or plurality of primers or probes comprises about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 35 nucleotides, about 40 nucleotides, about 45 nucleotides, and / or about 50 nucleotides. In some embodiments, the at least one or plurality of primers or probes comprises between about 20 nucleotides.

[0068] In some embodiments, the at least one or plurality of primers or probes can be a LNA enhanced primer or probe or combination thereof. For example, QIAseq Methyl panel, IDT targeted methyl sequencing, Agilent SureSelect Methyl-seq target can be used.

[0069] In some embodiments, when hybridizing with the one or more target sequence the at least one or plurality of primers or probes can for example comprise up to 3, up to 2 or up to 1 mismatch for every 20 base pairs.

[0070] In some embodiments, the at least one or plurality of probes are RNA probes, DNA probes, or a combination thereof.

[0071] In some embodiments, the panel comprises a substrate. The at least one or plurality of primers or probes can be affixed to the substrate, providing a convenient assay platform. They can also be provided for affixing to the substrate. In some embodiments, the substrate comprises a solid surface, optionally a chip, plate, beads, or slide. In some embodiments, the panel or the kit comprising the panel comprises two or more detection agents, each for detecting a target sequence described herein. For example, the panel can comprise a plurality of the probes or primers described herein. The panel can comprise one or more additional detection agents for detecting or measuring the levels of any one of target sequences described herein. In some embodiments, the panel is a microarray. In some embodiments, the plate is a 96 well plate. In some embodiments, the plate is a 1536 well plate. In some embodiments, the plate is a 384 well plate.

[0072] In some embodiments, the at least one or plurality of probes are immobilized on a substrate. In some embodiments, the substrate comprises a solid surface, a porous surface, or a combination thereof. In some embodiments, the substrate is a bead, a tube, a microtiter plate, a membrane, a gel, or a glass slide. In some embodiments, the at least one or plurality of primers or probes are immobilized spatially apart from each other on a substrate.

[0073] In some embodiments, the panel comprises at least one primer or probe per tissue type to be included in the panel. In some embodiments, the at least one or plurality of probes or primers comprises up to 50 probes or primers. In some embodiments, the at least one or plurality of probes or primers comprises up to 100 probes or primers. In some embodiments, the at least one or plurality of probes or primers comprises up to 150 probes or primers. In some embodiments, the at least one or plurality of probes or primers comprises up to 200 probes or primers. In some embodiments, the at least one or plurality of probes or primers comprises up to 300 probes or primers. In some embodiments, the at least one or plurality of probes or primers comprises up to 400 probes or primers. In some embodiments, the at least one or plurality of probes or primers comprises up to 500 probes or primers. In some embodiments, the at least one or plurality of probes or primers comprises up to 600 probes or primers. In some embodiments, the at least one or plurality of probes orprimers comprises up to 700 probes or primers. In some embodiments, the at least one or plurality of probes or primers comprises up to 800 probes or primers. In some embodiments, the at least one or plurality of probes or primers comprises up to 900 probes or primers. In some embodiments, the at least one or plurality of probes or primers comprises up to 1 ,000 probes or primers. In some embodiments, the at least one or plurality of probes or primers comprises up to 1 ,100 probes or primers. In some embodiments, the at least one or plurality of probes or primers comprises up to 1 ,200 probes or primers. In some embodiments, the at least one or plurality of probes or primers comprises up to 1 ,300 probes or primers. In some embodiments, the at least one or plurality of probes or primers comprises up to 1 ,400 probes or primers. In some embodiments, the at least one or plurality of probes or primers comprises up to 1 ,500 probes or primers. In some embodiments, the at least one or plurality of probes or primers comprises up to 1 ,600 probes or primers. In some embodiments, the at least one or plurality of probes or primers comprises up to 1 ,700 probes or primers. In some embodiments, the at least one or plurality of probes or primers comprises up to 1 ,800 probes or primers. In some embodiments, the at least one or plurality of probes or primers comprises up to 1 ,900 probes or primers. In some embodiments, the at least one or plurality of probes or primers comprises up to 2,000 probes or primers. In some embodiments, the at least one or plurality of probes or primers comprises up to 2,100 probes or primers. In some embodiments, the at least one or plurality of probes or primers comprises up to 2,200 probes or primers. In some embodiments, the at least one or plurality of probes or primers comprises up to 2,300 probes or primers. In some embodiments, the at least one or plurality of probes or primers comprises up to 2,400 probes or primers. In some embodiments, the at least one or plurality of probes or primers comprises up to 2,500 probes or primers. In some embodiments, the at least one or plurality of probes or primers comprises up to 2,600 probes or primers. In some embodiments, the at least one or plurality of probes or primers comprises up to 2,700 probes or primers. In some embodiments, the at least one or plurality of probes or primers comprises up to 2,800 probes or primers. In some embodiments, the at least one or plurality of probes or primers comprises up to 2,861 probes or primers.

[0074] In some embodiments, the at least one or plurality of probes comprises a detectable label.

[0075] In some embodiments, the at least one or plurality of primers or probes comprise a tag. In some embodiments, the tag comprises a common sequence, a unique molecular identifier, or an index tag.

[0076] Another aspect of the disclosure includes a method of identifying cell origin of cell free DNA molecules in a biological sample, the method comprising: a) determining a level of methylation of one or more target polynucleotide regions listed in Table 1 in the cell free DNA molecules; and b) identifying the cell origin of the cell free DNA molecules based on the level of methylation of the one or more target polynucleotide regions.

[0077] It is demonstrated herein that cell-free DNA isolated from perfusate samples from donor lungs that were subsequently transplanted has a profile of cellular origin different from donor lungs that were subsequently rejected.

[0078] Accordingly, in another aspect, provided herein is a method of predicting organ transplant outcome, the method comprising: (a) providing a biological sample; (b) isolating cell free DNA molecules from the biological sample; (c) determining a level of methylation for one or more target polynucleotide regions listed in Table 1 in the cell free DNA molecules; (d) identifying the cell origin of the cell free DNA molecules based on the methylation level of the one or more the one or more target polynucleotide regions, thereby predicting the transplant outcome.

[0079] In some embodiments, the organ transplant outcome comprises rejection of the organ. In some embodiments, the organ transplant outcome comprises transplant of the organ.

[0080] In some embodiments, the organ is a kidney, lung, liver, and / or pancreas transplant.

[0081] In some embodiments, the biological sample comprises a perfusate sample.

[0082] In a further aspect, provided herein is a method of diagnosing organ injury, the method comprising: providing a biological sample; isolating cell free DNA molecules from the biological sample; determining a level of methylation for one or more target polynucleotide regions listed in Table 1 in the cell free DNA molecules; identifying the cell origin of the cell free DNA molecules based on the methylation levelof the one or more the one or more target polynucleotide regions; thereby diagnosing organ injury.

[0083] The organ injury may be resulted from surgery, such as organ transplantation, trauma, infection, or any other causes. For example, the organ injury may be resulted from conditions such as trauma from external mechanical forces, medical conditions such as ischemia, infection, autoimmune diseases (e.g. inflammatory bowel disease, lupus) and cancer, surgery, toxic injury from drugs or other exogenous agents, and chronic diseases such as diabetes.

[0084] In some embodiments, the organ injury comprises a rejection of a transplant organ. In some embodiments, the organ injury comprises inflammation.

[0085] Each target polynucleotide region comprises at least one CpG motif. In some embodiments, the one or more target polynucleotide regions is a region identified as being hypo-methylated in a particular cell type. In some embodiments, the one or more target polynucleotide regions are a portion of any of the regions listed in Table 1 comprising at least one CpG motif. In some embodiments, the one or more target polynucleotide regions are up to about 1700 residues in length. The target polynucleotides can also be shorter, for example between 160 and 180 residues in length. Any length of the target polynucleotide that comprises at least one methylation site, e.g., at least one CpG (e.g., 2 residues), can be assessed. In some embodiments, the one or more target polynucleotide regions are at least 2 residues in length. In some embodiments, the one or more target polynucleotide regions are between 2 and about 1700 residues in length. In some embodiments, the one or more target polynucleotide regions are about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 residues in length. In some embodiments, the one or more target polynucleotide regions are about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 residues in length. In some embodiments, the one or more target polynucleotides are up to about 150 residues in length. In some embodiments, the one or more target polynucleotide regions are up to about 168 residues in length. In some embodiments, the one or more target polynucleotide regions are up to about 180 residues in length. In some embodiments, the one or more target polynucleotide regions are up to about 170 residues in length. In some embodiments, the one or more target polynucleotide regions are up to about 336 residues in length. In some embodiments, the one or more target polynucleotide regions are up to about 504 residues in length. In someembodiments, the one or more target polynucleotide regions are up to about 672 residues in length. In some embodiments, the one or more target polynucleotide regions are up to about 320 residues in length. In some embodiments, the one or more target polynucleotide regions are up to about 480 residues in length. In some embodiments, the one or more target polynucleotide regions are up to about 640 residues in length. In some embodiments, the one or more target polynucleotide regions are up to about 340 residues in length. In some embodiments, the one or more target polynucleotide regions are up to about 510 residues in length. In some embodiments, the one or more target polynucleotide regions are up to about 680 residues in length. In some embodiments, the one or more target polynucleotide regions are up to about 360 residues in length. In some embodiments, the one or more target polynucleotide regions are up to about 540 residues in length. In some embodiments, the one or more target polynucleotide regions are up to about 720 residues in length. In some embodiments, the portion of any of the regions listed in Table 1 comprises one CpG motif. In some embodiments, the portion of any of the regions listed in Table 1 comprises a plurality of CpG motifs. In some embodiments, the portion of any of the regions listed in Table 1 comprises between 1 to 70 CpG motifs. In some embodiments, the portion of any of the regions listed in Table 1 comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 CpG motifs. In some embodiments, the portion of any of the regions listed in T able 1 comprises about 1 , about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65 or about 70 CpG motifs.

[0086] In some embodiments, the biological sample is from a subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0087] The subject can be any subject comprising cell free DNA to be assessed. In some embodiments, the subject has or is suspected of having suffered organ damage. In some embodiments, the subject has received one or more organ transplant. In some embodiments, the subject is waiting to receive one or more organ transplant. In some embodiments, the one or more organ transplant is a kidney, lung, liver, and / or pancreas transplant. In some embodiments, the one or more organ transplant is a kidney, heart, intestine, lung, liver, vascular composite allograft, and / or pancreas transplant.

[0088] In some embodiments, the biological sample is taken from the subject prior to the subject receiving a transplanted organ. In some embodiments, the biological sample is taken from the subject after receiving a transplanted organ. In some embodiments, biological sample is taken from the subject immediately after receiving a transplanted organ. In some embodiments, the biological sample is taken from the subject 24 hours after receiving a transplanted organ.

[0089] In some embodiments, the methods described herein are performed one or more times after the subject has received a transplanted organ. In some embodiments, the methods described herein are performed one or more times after the subject has suffered or is suspected to have suffered organ damage.

[0090] In some embodiments, the biological sample is any biological sample comprising cell free DNA. In some embodiments, the biological sample is a tissue sample.

[0091] In some embodiments, the biological sample is a plasma sample. In some embodiments, the biological sample is a serum sample. In some embodiments, the biological sample is an organ or tissue perfusate sample. In some embodiments, the perfusate sample is from perfusion of a lung. In some embodiments, the biological sample is a urine sample. In some embodiments, the biological sample is sputum. In some embodiments, the biological sample is a bronchoalveolar washing sample. In some embodiments, the biological sample comprises a sample from a pleural effusion. In some embodiments, the biological sample is a cerebrospinal fluid sample. In some embodiments, the determining of the level methylation comprises contacting the cell free DNA molecules with the one or more primers or the panel described herein. In some embodiments, the determining of the level methylation comprises contacting the cell free DNA molecules with the one or more probes or the panel described herein e.g., a microarray.

[0092] In some embodiments, the determining of the level methylation comprises subjecting the biological sample to unbiased whole genome bisulfite sequencing.

[0093] In some embodiments, the determining of the level of the methylation comprises amplifying the one or more target polynucleotide regions. Any suitable reagents for amplifying the targets or a library of the targets can be used. In someembodiments, the reagent for amplifying the targets or a library of the targets can comprise an enzyme, e.g., an enzyme to be used in a polynucleotide amplification reaction. Exemplary polynucleotide amplification reactions include polymerase chain reaction (PCR).

[0094] In some embodiments, the one or more target polynucleotide regions comprise one CpG motif. In some embodiments, the one or more target polynucleotide regions comprise a plurality of CpG motifs. In some embodiments, the one or more target polynucleotide regions comprise between 1 to 70 CpG motifs. In some embodiments, the one or more target polynucleotide regions comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 CpG motifs. In some embodiments, the one or more target polynucleotide regions comprise about 1 , about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65 or about 70 CpG motifs.

[0095] In some embodiments, determining of the level of the methylation comprises isolating the one or more target polynucleotide regions.

[0096] In some embodiments, the determining of the level of the methylation comprises preparing a library of a plurality of the one or more target polynucleotide regions. Any suitable reagents for preparing a library of the targets can be used.

[0097] In another embodiment, determining of the level of the methylation can further comprise purifying the target polynucleotide regions, a library of the target polynucleotide regions, amplified target polynucleotide regions or a library of amplified target polynucleotide regions.

[0098] The level of methylation of target polynucleotide regions can be determined using any suitable methods and / or reagents, which are known in the art. In some embodiments, the level of methylation of target polynucleotide regions can be determined using mass spectrometry, methylation-specific PCR (MSP), methylationsensitive sequencing, e.g., bisulfite sequencing, the HpaTI tiny fragment Enrichment by ligation-mediated PCR assay (HELP Assay), nanopore, single molecule real time (SMRT) sequencing, Glal hydrolysis and ligation adapter dependent PCR assay (GLAD-PCR assay), methylated DNA immunoprecipitation (MeDIP or mDiP), pyrosequencing, molecular break light assay for DNA adenine methyltransferase activity, methyl sensitive Southern blotting or high resolution Melt (HRM) analysis.

[0099] In some embodiments, the level of methylation of target polynucleotide regions can be determined using a chemical agent, e.g., bisulfite or sodium bisulfite. In some embodiments, the level of methylation of target polynucleotide regions can be determined using a biological agent, e.g., an antibody.

[0100] In some embodiments, the determining the level of methylation of the one or more target polynucleotide regions comprises using methylation-sensitive sequencing. In some embodiments, the methylation-sensitive sequencing comprises bisulfite sequencing. Bisulfite conversion is a method that uses bisulfite to determine the methylation pattern of DNA. DNA methylation is a biochemical process involving the addition of a methyl group to the cytosine or adenine DNA nucleotides. In bisulfite conversion, target nucleic acids are first treated with bisulfite reagents that specifically convert un-methylated cytosines to uracils while having no impact of methylated cytosine.

[0101] Any suitable format of methylation- sensitive sequencing can be used. Examples of such methylation-sensitive sequencing are known in the art. For example, the methylation-sensitive sequencing can be conducted with a format selected from Maxam-Gilbert sequencing, a chain-termination method, shotgun sequencing, bridge PCR, single-molecule real-time sequencing, ion semiconductor (ion torrent sequencing), sequencing by synthesis, paired end sequencing, sequencing by ligation (SOLiD sequencing), chain termination (Sanger sequencing), massively parallel signature sequencing (MPSS), polony sequencing, 454 pyrosequencing, Illumina (Solexa) sequencing, DNA nanoball sequencing, heliscope single molecule sequencing, single molecule real time (SMRT) sequencing, nanopore DNA sequencing, tunnelling currents DNA sequencing, sequencing by hybridization, sequencing with mass spectrometry, microfluidic Sanger sequencing, a microscopybased technique, RNAP sequencing, and in vitro virus high-throughput sequencing.

[0102] Other suitable methods to determine the level of methylation of the one or more target polynucleotide regions may be used. For example, the cell free DNA may be digested with one or more methylation specific restriction enzymes. If a target polynucleotide region is methylated in a cfDNA molecule, then it may not be amplified by two primers flanking the cleaved site. Therefore, the level of methylation at the target polynucleotide region may be quantified, for example, by quantitative real-timePCR (qRT-PCR) of the target polynucleotide region, by comparing results from a digested biological sample to a control.

[0103] In some embodiments, the determining the level of methylation of the one or more target polynucleotide regions comprises subjecting the cell free DNA molecules to methylation specific restriction digest by one or more methylationsensitive restriction enzymes. In some embodiments, the determining the level of methylation of the one or more target polynucleotide regions comprises performing quantitative PCR on methylation-sensitive restriction enzyme digested cfDNA molecules. In some embodiments, the quantitative PCR is performed using a probe or primer disclosed herein.

[0104] Level of methylation of one or more or each of the target polynucleotide regions can be determined. In some embodiments, the level of methylation of each of the target polynucleotide regions can be used to obtain a methylation metric, e.g., in the form of average methylation frequency, methylation haplotype load, unmethylation haplotype load, percent discordant reads, or a combination thereof.

[0105] For example, during bisulfite treatment, methylated cytosines are protected, while unmethylated cytosines are converted to uracils, which are then replaced by thymines during amplification. For cytosines on the plus strand this manifests as C-to-T change when mapped to the plus-strand of the genome and for cytosines on the minus strand this manifests as G-to-A changes. Therefore, counting the number of sequencing reads with C within identified CpGs can indicate a level of methylation. Where a region contains more than one CpG, an average methylation value may be calculated from the methylation values of all CpG’s within that region.

[0106] In some embodiments, the one or more target polynucleotide regions comprise one CpG motif. In some embodiments, the one or more target polynucleotide regions comprise a plurality of CpG motifs. In some embodiments, the one or more target polynucleotide regions comprise between 1 to 70 CpG motifs. In some embodiments, the one or more target polynucleotide regions comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 CpG motifs. In some embodiments, the one or more target polynucleotide regions comprise about 1 , about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65 or about 70 CpG motifs.

[0107] In some embodiments, the level of methylation of each of the one or more target polynucleotide regions is used to calculate a methylation metric.

[0108] In some embodiments, the identification of the cell origin comprises comparison of the methylation metric for each of the target polynucleotide regions to a reference methylation metric associated with a particular cell type. In some embodiments, the methylation metric for each of the target polynucleotide regions is an average methylation rate. In some embodiments, the reference methylation metric is an average methylation rate. In some embodiments, the methylation metric is a raw CpG methylation values.

[0109] In some embodiments, the reference methylation metric is calculated from a cell-type specific methylation atlas.

[0110] In some embodiments, the cell free DNA molecules are present in a mixture of cell free DNA molecules from same cell origin. In some embodiments, the cell free DNA molecules are present in a mixture of cell free DNA molecules from different cell origins.

[0111] In some embodiments, the method further comprises determining the contribution level of the cell free DNA molecules from different cell origins in a mixture of cell free DNA molecules. In some embodiments, the level is expressed as a percentage of the total cell free DNA in the mixture.

[0112] In some embodiments, the contribution level of the cell free DNA molecules from different cell origins in a mixture of cell free DNA molecules is determined using a similarity algorithm, a methylation metric for each of the target polynucleotide regions and a reference methylation metric. In some embodiments, the similarity algorithm is non-negative least squares or matrix decomposition. In some embodiments, the methylation metric for each of the target polynucleotide regions and the reference methylation metric is an average methylation rate.

[0113] In some embodiments, the subject is a transplant recipient, and the cell free DNA molecules are not subject cell free DNA. In some embodiments, the cell free DNA molecules is donor-derived cell free DNA (e.g., from an organ donor). In some embodiments, the genotype of the subject receiving the transplant is known. In some embodiments, the subject has received an organ from a donor and the organ donor genotype is known.

[0114] In some embodiments, any method described herein can be performed prior to or subsequent to one or more other tests (e.g., a test for identifying presence of donor derived DNA or a single nucleotide variant test).

[0115] In some embodiments, the method described herein comprises use of one or more primer or probe comprises the nucleotide sequence of any one of SEQ ID NOs: 1 -2921 or least 20 consecutive residues of any thereof, or a panel comprising a plurality of the at least one primer or probe.

[0116] Another aspect of the disclosure includes a kit comprising the panel described herein e.g., a plurality of the primers or probes as described herein. In some embodiments, the kit further comprises a substrate. In some embodiments, the kit comprises instructions for use. In some embodiments, the kit comprises one or more containers e.g., vials or tubes. In some embodiments, the kit comprises a one or more reagents for amplification of target polynucleotide regions or libraries. In some embodiments, the substrate is (such as a microarray) for capture probes for detecting of amplified nucleic acids. In some embodiments, the substrate is a part of a bead, a tube, a microtiter plate, a membrane, a gel, or a glass slide. In some embodiments, the probes are immobilized spatially apart from each other on a substrate.

[0117] Also contemplated herein are uses based on the methods described herein.

[0118] It should also be understood that, in certain methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise.

[0119] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.ExamplesExample 1

[0120] Donor-derived cell-free DNA (ddcfDNA) has been shown to be associated with post-transplant lung conditions such as rejection and infection. The prevailing hypothesis is that this is lung-derived cfDNA from the donor's lung injury. However, the cellular origin of ddcfDNA has been poorly characterized, which limits the utility of ddcfDNA detection. Herein, the cell origin of ddcfDNA in the immediate post-transplant period is profiled using cfDNA methylation.

[0121] Methods: A total of N=10 transplant cases were used in this singlecenter, retrospective cohort study. Plasma samples were collected at 24h posttransplant. Healthy patient samples (N=2) were also collected. The cfDNA was extracted and the enzymatic methyl sequencing method was used for library preparation, followed by Illumina sequencing. Cell of origin was predicted from the samples using publicly available tissue atlases.

[0122] Results: Cell of origin analysis performed from methylation sequencing detected 39 cell origins (Fig. 10). Tissues with an origin of less than 5% were removed (FIG. 10). From these results, it is observed that the majority of cfDNA was of granulocyte origin (51.3 + / - 10.7%), monocyte -i-macrophage origin (10.2 + / - 3.5%), and endothelial origin (9.8 + / - 2.9%). Lung epithelium alveolar origin (0.5 + / - 0.4%) and lung epithelium bronchus origin (0.4 + / - 0.5%) were detected.

[0123] In this cohort, the majority of cfDNA released in the early transplant period is of leukocytic origin, representing either recipient inflammation and / or passenger donor leukocyte apoptosis. The endothelium signal may represent pulmonary endothelial damage of reperfusion injury.Example 2:Identification of sets of CpGs that have consistent methylation patterns in different cell / tissue types for profiling of cfDNA samples

[0124] Loyfer et al., 2023 (“A DNA methylation atlas of normal human cell types.” Nature vol. 613,7943 (2023): 355-364) describes using methylation data from multiple samples from 39 tissue types to generate a methylation atlas.

[0125] Regions used in Loyfer et al., 2023, were large and may have lower mappability for ~168bp cfDNA fragments. Therefore, a proprietary methylation callingpipeline was developed to find small hypo-methylated regions with high mappability using the GRCh38- human reference genome: CpGs that are closely spaced (<1 OObp apart) in small regions were identified, and ones that are specifically hypomethylated were identified in each tissue type (FIG. 2).

[0126] Regions of similar CpG methylation can be cell-type specific and useful to predict the cell-type composition of a heterogeneous sample; for example, whole plasma cfDNA. FIG. 1 B shows specific CpG hypomethylated regions in alveolar cells from the lung.

[0127] The hypomethylated cell-type specific regions using the proprietary methylation calling pipeline were validated using a second publicly available atlas (FIG. 3). Only tissues with a contribution of at least 5% are included in FIG. 3.Example 3 - Developing a panel for determining cell origin from cell free DNA

[0128] A custom targeted methylation panel suitable for cfDNA analysis was designed based on 15 cell types: Kidney-Epithelial, Blood-B, Pancreas-Alpha, Pancreas-Beta, Blood-Monocyte+Lung-Macrophage, Blood-NK, Endothelial, Blood-T, Lung-Bronchus-Epithelial, Lung-Alveolar-Epithelial, Blood-Granulocytes, Pancreas- Delta, Liver-Hepatocytes, Pancreas-Acinar, Pancreas-Duct. Different cell types can be added or removed as needed. The panel can use bisulfite-treated samples and is strand-specific and provides unique molecular identifiers (Fig. 6). The panel uses a single primer to maximize sensitivity for small fragments. The small fragments make it so that the fragments are closer together and more CpG sites are easier to target, which allows for a more accurate methylation value to be calculated.

[0129] Primers were designed (SEQ ID NOs: 1 -2921 ), which cover CpG sites (see e.g., Table 1 ). Primers were designed by Qiagen for the QIAseq targeted methylation panel kit which includes unique molecular identifiers (FIG. 6).

[0130] Libraries were generated from cfDNA samples using the panel. The resultant libraries were sequenced using paired-end sequencing. The sequencing data was processed to determine methylation levels, represented by methylation values.

[0131] Based on the methylation values, cell origin can be deconvoluted using a technique such as non-negative least squares or matrix decomposition.

[0132] Results:

[0133] FIG. 7 shows an output of a panel incorporating the cell-origin methylation patterns. It is able to predict the cell origins of cfDNA from post-transplant plasma, perfusate, and lung tissue with the expected cell origins.

[0134] The panel was validated using sorted cell types with matched EM-seq (FIGs. 8-9). FIG. 8 shows the prediction of cell origin between the panel and EM-seq (non-panel). The panel is more sensitive as it is able to have massively increased sequencing coverage of the genomic loci that cover the regions identified using our methylation calling pipeline. FIG. 9 summarizes the increased coverage between sequencing all of the cfDNA in a sample by EM-seq vs the panel.Example 4 - Predicting transplant outcome

[0135] In ex-vivo lung perfusion, human lungs are perfused on a close loop and ventilated. DNAses- free perfusate solution is used, which may better preserve any released DNA. Perfusate contains cfDNA. Perfusate has cfDNA fragments with the expected sizes but also large fragments that could be from NETs or undegraded DNA (Fig. 5). Library preparation involves removing large fragments and small fragments.

[0136] FIG. 7 shows that in a cohort of perfusate cfDNA samples collected following EVLP, there is a difference in cell-origin pattern between the lungs transplanted after EVLP and the lungs rejected for transplant after EVLP. The amount of monocyte origin and granulocyte origin cfDNA appears to be driving this difference (Figure 7, bottom). A signature / score can be calculated by subtracting the monocyte origin (%) from the granulocyte origin (%) (FIG. 7, top). This signature can predict whether a lung will be transplanted or rejected with an area under the curve of ~0.8 (FIG. 7, top).Table 1. Regions of hypomethylation (Genome assembly GRCh38)

[0137] While the present application has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the application is not limited to the disclosed examples. To the contrary, the application is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0138] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Specifically, the sequences associated with each accessionnumbers provided herein including for example accession numbers and / or biomarker sequences (e.g. protein and / or nucleic acid) provided in the Tables or elsewhere, are incorporated by reference in its entirely.

[0139] The scope of the claims should not be limited by the preferred embodiments and examples but should be given the broadest interpretation consistent with the description as a whole.

Claims

CLAIMS1 . A method of identifying cell origin of cell free DNA molecules in a biological sample, the method comprising: a) determining a level of methylation for one or more target polynucleotide regions listed in Table 1 in the cell free DNA molecules; and b) identifying the cell origin of the cell free DNA molecules based on the methylation level of the one or more the one or more target polynucleotide regions.

2. A method of diagnosing organ injury, the method comprising: a) providing a biological sample; b) isolating cell free DNA molecules from the biological sample; c) determining a level of methylation for one or more target polynucleotide regions listed in Table 1 in the cell free DNA molecules; d) identifying cell origin of the cell free DNA molecules based on the methylation level of the one or more the one or more target polynucleotide regions, thereby diagnosing organ injury.

3. The method of claim 2, wherein the organ injury comprises a rejection of a transplant organ.

4. The method of claim 2, wherein the organ injury comprises inflammation.

5. The method of claim 2, wherein the organ injury comprises organ trauma.

6. The method of any one of claims 1 -5, wherein the biological sample is a plasma sample.

7. The method of any one of claims 1 -5, wherein the biological sample is a perfusate sample.

8. The method of any one of claims 1 -5, wherein the biological sample is from a subject, optionally a human.

9. The method of claim 8, wherein the subject has received one or more organ transplant.

10. The method of claim 9, wherein the one or more organ transplant is a kidney, lung, liver, and / or pancreas transplant.1 1 . The method of claim 8, wherein genotype of the subject is known.

12. The method of claim 8, wherein the subject is a transplant recipient and the cell free DNA molecules detected are not subject cell free DNA.

13. The method claim 8, wherein the subject has received an organ from a donor and the organ donor genotype is known14. A method of predicting a transplant outcome, comprising: a) providing a biological sample; b) isolating cell free DNA molecules from the biological sample; c) determining a level of methylation for one or more target polynucleotide regions listed in Table 1 in the cell free DNA molecules; d) identifying the cell origin of the cell free DNA molecules based on the methylation level of the one or more the one or more target polynucleotide regions, thereby predicting the transplant outcome.

15. The method of claim 14, wherein the transplant outcome comprises rejection of a transplant organ.

16. The method of claim 14, wherein the transplant outcome comprises transplant of a transplant organ.

17. The method of any one of claims 14-16, wherein the transplant organ is a kidney, lung, liver, and / or pancreas transplant.

18. The method of any one of claims 14-17, wherein the biological sample comprises a perfusate sample.

19. The method of any one of claims 1 to 18, wherein the determining of the level of methylation comprises contacting the cell free DNA molecules with at least oneprimer or probe complementary to one or more target sequence, wherein each of the one or more target sequence is comprised in a region listed in Table 1 .

20. The method of claim 19, wherein the one or more primer or probe comprises the nucleotide sequence of any one of SEQ ID NOs: 1 -2921 or least 20 consecutive residues of any thereof, or a panel comprising a plurality of the at least one primer or probe.21 . The method of any one of claims 1 to 20, wherein the determining of the level of methylation comprises amplifying the one or more target polynucleotide regions.

22. The method of any one of claims 1 to 21 , wherein the determining of the level of methylation of the one or more target polynucleotides comprises using methylation-sensitive sequencing.

23. The method of any one of claims 1 to 20, wherein the determining of the level of methylation comprises subjecting the cell free DNA molecules to methylationspecific restriction digest by one or more methylation-sensitive restriction enzymes.

24. The method of claim 23, wherein the determining the level of methylation of the one or more target polynucleotide regions comprises performing quantitative PCR on methylation-sensitive restriction enzyme digested cell free DNA molecules.

25. The method of claim 24, wherein the quantitative PCR is performed using one or more primer or probe comprises the nucleotide sequence of any one of SEQ ID NOs: 1 -2921 or least 20 consecutive residues of any thereof.

26. The method of one of claims 1 to 25, wherein a methylation metric is obtained based on the level of methylation of each of the one or more target polynucleotide regions.

27. The method of claim 26, wherein the identification of the cell origin comprises comparison of the methylation metric for each of the target polynucleotide regions to a reference methylation metric associated with a cell type.

28. The method of claim 26 or 27, wherein the methylation metric for each of the target polynucleotide regions is an average methylation rate.

29. The method of claim 28, wherein the reference methylation metric is an average methylation rate.

30. The method of any one of claims 27-29, wherein the reference methylation metric is calculated from a cell-type specific methylation atlas.31 . The method of any one of claims 1 to 30, wherein the cell free DNA molecules are present in a mixture of cell free DNA molecules from the same cell origin or from different cell origins.

32. The method of claim 31 , the method further comprising determining the contribution level of different cell origins in a mixture of cell free DNA molecules.

33. The method of claim 32, wherein the contribution level of different cell origin in a mixture of cell free DNA molecules is determined using a similarity algorithm and a methylation metric for each of the target polynucleotide regions and a reference methylation metric.

34. The method of claim 33, wherein the similarity algorithm is non-negative least squares.

35. The method of claim 33 or 34, wherein the methylation metric for each of the target polynucleotide regions and the reference methylation metric are an average methylation rate.

36. At least one primer or probe complementary to one or more target sequence, wherein each of the one or more target sequence is comprised in a region listed in Table 1 .

37. The at least one primer or probe of claim 36 comprising the nucleotide sequence of any one of SEQ ID NOs: 1 -2921 , or at least 20 consecutive residues of any thereof.

38. A composition comprising the one or more primer or probe of claim 36 or 37.

39. A panel comprising a plurality of the one or more primers or probes of claim36 or 37 or the composition of claims 38.

40. A kit comprising the panel of claim 39 or a plurality of the one or more primer or probe of claim 36 or 37 or the composition of claim 38.41 . The panel of claim 39 or the kit of claim 40, wherein the panel further comprises a substrate onto which the at least one or plurality of primers or probes is affixed.

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