Cancer diagnosis and treatment response screening with fragmentomics and methylation analysis

Abstract

Disclosed herein are methods of determining a cell free DNA (cfDNA) methylation profile and a cfDNA fragmentation profile in a single DNA sample from a subject. In one aspect, the disclosed methods relate to identifying methylated cytosine in a target nucleic acid from the DNA sample and obtaining sequenced fragments from the same sample; mapping the sequenced fragments to a genome to obtain genomic intervals of mapped sequences; and, analyzing the genomic intervals of mapped sequences to determine cfDNA fragment lengths and determining the cfDNA fragmentation profile using the lengths.

Description

PATENT ATTORNEY DOCKET NO. DELFI2170-1WOCANCER DIAGNOSIS AND TREATMENT RESPONSE SCREENING WITH FRAGMENTOMICS AND METHYLATION ANALYSISCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present invention claims priority to U.S. Provisional Patent Application No. 63 / 697.990, titled CANCER DIAGNOSIS AND TREATMENT RESPONSE SCREENING WITH FRAGMENTOMICS AND METHYLATION ANALYSIS, filed September 23, 2024, which is hereby incorporated by reference herein in its entirety.FIELD OF THE INVENTION

[0002] The present invention relates generally to DNA methylation analysis and fragmentomics, and more specifically to combining methylation sequencing methods that utilizes enzymatic conversion of methylated cytosine and fragmentomic signals to analyze cell free DNA (cfDNA).BACKGROUND INFORMATION

[0003] Late diagnosis for cancer, where treatments are less effective, is linked to high morbidity7and mortality7of human cancers. Cell-free DNA analysis has been described as potential to provide new avenues for early diagnosis. There is a need for biomarkers analysis of cfDNA that can be used for early disease diagnosis and monitoring of treatment response to improve mortality and morbidity.

[0004] Epigenetic modifications are linked to various diseases, such as cancer, neurological disorders, and cardiovascular diseases. A common ty pe of epigenetic modification is DNA methylation. DNA is typically methylated at cytosine residues in CpG. Altered methylation profile are implicated in early disease stages, progression and treatment response.

[0005] The current gold standard and most widely used method for DNA methylation analysis is bisulfite sequencing (BS) and its related methods, such as TET (ten eleven translocation)- assisted bisulfite sequencing (TAB-Seq) and oxidative bisulfite sequencing (oxBS). These methods utilize bisulfite treatment to convert unmethylated cytosine to uracil while leaving methylated cytosines intact. The modification information of each cytosine can be inferred at a single base resolution (where the transition of C to T provides the location of the unmethylated cytosine), which reads uracil as thymine, using PCR amplification of the bisulfite-treated DNA. However, the use of bisulfite sequencing has disadvantages. Bisulfite treatment is a harsh chemical reaction, which degrades more than 90% of the DNA due to depurination under the required acidic and thermal conditions. This degradation severely' limits its application to low-11623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WO input samples, such as clinical samples including circulating cell-free DNA and single-cell sequencing. Additionally, this degradation excludes further analysis of DNA on the same sample.

[0006] Another DNA analysis method that has been implicated in disease diagnosis, progression, and treatment assessment is the study of DNA or RNA fragments present in biological samples. The study of the implication and the characteristics of these fragments is referred to as fragmentomics. Fragmentomics has been employed in the study of diseases such as cancer, infectious disease and autoimmune disorders and provides a valuable framework for understanding and utilizing the role of nucleic acid fragments in disease assessment and treatment. Combining fragmentomic signals and DNA methylation signals can provide a more sensitive and sophisticated method for screening and diagnosing cancer. There is a need for a combination analysis that will circumvent the limitations of the bisulfite sequencing method and allow methylation and fragmentomics analysis on the same sample.SUMMARY

[0007] The present disclosure relates to analysis that combines methylation sequencing methods that utilize enzymatic conversion of methyl cytosine with fragmentomics analysis to determine DNA methylation profile and fragmentation profiles in the same DNA sample. TET-assisted pyridine borane sequencing (TAPS) is one of the DNA methylation analysis technologies that utilizes such gentle enzymatic conversion.

[0008] In one aspect, the present disclosure provides a method of determining a cell free DNA (cfDNA) methylation profile and a cfDNA fragmentation profile in a single DNA sample from a subject including: (i) identifying 5-methylcytosine (5mC) in a target nucleic acid from the DNA sample including: a) modifying target nucleic acid in the DNA sample including: 1) adding a blocking group to 5-hydroxymethylcytosine (5hmC) in the DNA sample; 2) converting the 5mC in the DNA sample to 5-carboxylcytosine (5caC) and / or 5-formylcytosine (5fC); and 3) converting the 5caC and / or 5fC to dihydrouracil (DHU) to provide a modified DNA sample including a modified target nucleic acid; b) preparing sequencing libraries; and c) generating a sequence of the modified target nucleic acid; wherein a cytosine (C) to thymine (T) transition in the sequence of the modified target nucleic acid compared to the target nucleic acid provides a location of a 5mC in the target nucleic acid; and subjecting the sequencing libraries obtained from step (i) to whole genome sequencing to obtain sequenced fragments; mapping the sequenced fragments to a genome to obtain genomic intervals of mapped sequences; and, analyzing the21623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WO genomic intervals of mapped sequences to determine cfDNA fragment lengths and determining the cfDNA fragmentation profile using the lengths.

[0009] In one aspect, the present disclosure provides a method of determining a cell free DNA (cfDNA) methylation profile and a cfDNA fragmentation profile in a single DNA sample from a subject including: (i) identifying methylated cytosine in a target nucleic acid from the DNA sample including: a) modifying target nucleic acid in the DNA sample including performing enzymatic conversion of methylated cytosine; b) prepare sequencing libraries; and c) generating a sequence of the modified target nucleic acid; wherein a cytosine conversion in the sequence of the modified target nucleic acid compared to the target nucleic acid provides a location of a methyl cytosine in the target nucleic acid; and (ii) subjecting the sequencing libraries obtained from step (i) to whole genome sequencing to obtain sequenced fragments; mapping the sequenced fragments to a genome to obtain genomic intervals of mapped sequences; and, analyzing the genomic intervals of mapped sequences to determine cfDNA fragment lengths and determining the cfDNA fragmentation profile using the lengths.

[0010] In one aspect, the present disclosure provides a method of diagnosing cancer or screening for early detection of cancer in a subject including: (i) determining a cell free DNA (cfDNA) methylation profile including identifying 5-methylcytosine (5mC) in a target nucleic acid in a DNA sample from the subject including: a) modifying target nucleic acid in the sample including: 1) adding a blocking group to 5 -hydroxy methylcytosine (5hmC) in the DNA sample; 2) converting the 5mC in the DNA sample to 5-carboxylcytosine (5caC) and / or 5-formylcytosine (5fC); and 3) converting the 5caC and / or 5fC to dihydrouracil (DHU) to provide a modified DNA sample including a modified target nucleic acid; b) preparing sequencing libraries; and c) generating a sequence of the modified target nucleic acid; wherein a cytosine (C) to thymine (T) transition in the sequence of the modified target nucleic acid compared to the target nucleic acid provides a location of a 5mC in the target nucleic acid; (ii) determining a cfDNA fragmentation profile in the DNA sample from the subject including: a) subjecting the sequencing libraries obtained from step (i) to whole genome sequencing to obtain sequenced fragments; b) mapping the sequenced fragments to a genome to obtain genomic intervals of mapped sequences; and, c) analyzing the genomic intervals of mapped sequences to determine cfDNA fragment lengths and determining the cfDNA fragmentation profile using the lengths; and (ii) diagnosing the subject with cancer if the level of cfDNA methylation in the subject is altered as compared to the level of cDNA methylation in a healthy subject, the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject, or the level of cfDNA methylation31623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WO in the subject is altered as compared to the level of cDNA methylation in a healthy subject and the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject.

[0011] In one aspect, the present disclosure provides a method of diagnosing cancer or screening for early detection of cancer in a subject comprising: (i) determining a cell free DNA (cfDNA) methylation profile comprising identifying 5-methylcytosine (5mC) in a target nucleic acid in a DNA sample from the subject comprising: a) modifying target nucleic acid in the sample comprising: 1) adding a blocking group to 5-hydroxymethylcytosine (5hmC) in the DNA sample; 2) converting the 5mC in the DNA sample to 5 -carboxylcytosine (5caC) and / or 5-formylcytosine (5fC); and 3) converting the 5caC and / or 5fC to dihydrouracil (DHU) to provide a modified DNA sample comprising a modified target nucleic acid; b) preparing sequencing libraries; and c) generating a sequence of the modified target nucleic acid; wherein a cytosine (C) to thymine (T) transition in the sequence of the modified target nucleic acid compared to the target nucleic acid provides a location of a 5mC in the target nucleic acid; (ii) determining a cfDNA fragmentation profile in the DNA sample from the subject comprising: a) subjecting the sequencing libraries obtained from step (i) to whole genome sequencing to obtain sequenced fragments; b) mapping the sequenced fragments to a genome to obtain genomic intervals of mapped sequences; and, c) analyzing the genomic intervals of mapped sequences to determine cfDNA fragment lengths and determining the cfDNA fragmentation profile using the lengths; and (iii) diagnosing the subject with cancer if the methylation profile is characteristic of a methylation profile in a subject with cancer, the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject, or the methylation profile is characteristic of a methylation profile in a subject with cancer and the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subj ect.

[0012] In one aspect, the present disclosure provides a method of monitoring cancer treatment response in a subject in need thereof, including: (i) determining a cell free DNA (cfDNA) methylation profile including identifying 5-methylcytosine (5mC) in a target nucleic acid in a DNA sample from the subject including: a) modifying target nucleic acid in the DNA sample including: 1) adding a blocking group to 5-hydroxymethylcytosine (5hmC) in the DNA sample; 2) converting the 5mC in the DNA sample to 5 -carboxylcytosine (5caC) and / or 5-formylcytosine (5fC); and 3) converting the 5caC and / or 5fC to dihydrouracil (DHU) to provide a modified DNA sample including a modified target nucleic acid; b) preparing sequencing libraries; and c) generating a sequence of the modified target nucleic acid; wherein a cytosine (C) to thymine (T)41623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WO transition in the sequence of the modified target nucleic acid compared to the target nucleic acid provides a location of a 5mC in the target nucleic acid; (ii) determining a cfDNA fragmentation profile in the DNA sample from the subject including: a) subjecting the sequencing libraries obtained from step (i) to whole genome sequencing to obtain sequenced fragments; b) mapping the sequenced fragments to a genome to obtain genomic intervals of mapped sequences; and c) analyzing the genomic interv als of mapped sequences to determine cfDNA fragment lengths and determining the cfDNA fragmentation profile using the lengths; and (iii) determining the subject still has cancer if the level of cfDNA methylation in the subject is altered as compared to the level of cDNA methylation in a healthy subject, the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject, or the level of cfDNA methylation in the subject is altered as compared to the level of cDNA methylation in a healthy subject and the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject.

[0013] In one aspect, the present disclosure provides a method of monitoring cancer treatment response in a subject in need thereof, including: (i) determining a cell free DNA (cfDNA) methylation profile including identifying 5-methylcytosine (5mC) in a target nucleic acid in a DNA sample from the subject including: a) modifying target nucleic acid in the DNA sample including: 1) adding a blocking group to 5-hydroxymethylcytosine (5hmC) in the DNA sample; 2) converting the 5mC in the DNA sample to 5-carboxylcytosine (5caC) and / or 5-formylcytosine (5fC); and 3) converting the 5caC and / or 5fC to dihydrouracil (DHU) to provide a modified DNA sample including a modified target nucleic acid; b) preparing sequencing libraries; and c) generating a sequence of the modified target nucleic acid; wherein a cytosine (C) to thymine (T) transition in the sequence of the modified target nucleic acid compared to the target nucleic acid provides a location of a 5mC in the target nucleic acid; (ii) determining a cfDNA fragmentation profile in the DNA sample from the subject including: a) subjecting the sequencing libraries obtained from step (i) to whole genome sequencing to obtain sequenced fragments; b) mapping the sequenced fragments to a genome to obtain genomic intervals of mapped sequences; and c) analyzing the genomic interv als of mapped sequences to determine cfDNA fragment lengths and determining the cfDNA fragmentation profile using the lengths; and (iii) determining the subject still has cancer if the methylation profile is characteristic of a methylation profile in a subject with cancer, the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject, or the methylation profile is characteristic of a methylation profile51623885965.1PATENTATTORNEY DOCKET NO. DELFI2170-1WO in a subject with cancer and the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subj ect.

[0014] In some embodiments, the step of generating the sequence of the modified target nucleic acid comprises one or more of chain termination sequencing, microarray, high-throughput sequencing, and restriction enzyme analysis.

[0015] In some embodiments, the percentages of a T at each transition location provide a quantitative level of 5mC at each location in the target nucleic acid.

[0016] In some embodiments, the mapped sequences comprise tens or hundreds to thousands of genomic intervals.

[0017] In some embodiments, the genomic intervals are non-overlapping.

[0018] In some embodiments, the genomic intervals each comprise thousands to millions of base pairs.

[0019] In some embodiments, a cfDNA fragmentation profile is determined within each genomic intervals.

[0020] In some embodiments, the cfDNA fragmentation profile comprises a median fragment size.

[0021] In some embodiments, the cfDNA fragmentation profile comprises a fragment size distribution.

[0022] In some embodiments, the cfDNA fragmentation profile comprises a ratio of small cfDNA fragments to large cfDNA fragments in said windows of mapped sequences.

[0023] In some embodiments, the cfDNA fragmentation profile comprises the sequence coverage of small cfDNA fragments in genomic intervals across the genome.

[0024] In some embodiments, the cfDNA fragmentation profile comprises the sequence coverage of large cfDNA fragments in genomic intervals across the genome.

[0025] In some embodiments, the cfDNA fragmentation profile comprises the sequence coverage of small and large cfDNA fragments in genomic intervals across the genome.

[0026] In some embodiments, methylation profiles characteristic of cancer are determined using prior machine learning in a subject with cancer compared to methylation profiles in a healthy subject.

[0027] In some embodiments, methylation profiles characteristic of cancer are determined based on methylation profiles described in the scientific literature.61623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WO

[0028] In some embodiments, methylation profiles characteristic cancer are determined based on a combination of using prior machine learning in a subject with cancer compared to methylation profiles in a healthy subject and methylation profiles described in the scientific literature.

[0029] In some embodiments, the methylation profiles characteristic of cancer are determined by increased level of cfDNA methylation in the subject as compared to level of cfDNA methylation in a healthy subject.

[0030] In some embodiments, the methylation profiles characteristic of cancer incorporate the position of methylated cytosine with respect to the ends of cfDNA fragments.

[0031] In some embodiments, genome coverage is about 9x to 0. lx.

[0032] In some embodiments, the cancer is selected from the group consisting of: colorectal cancer, lung cancer, breast cancer, gastric cancer, pancreatic cancer, bile duct cancer, and ovarian cancer.

[0033] In some embodiments, a therapeutic treatment suitable for treatment of the cancer is administered to the subject if the subject is diagnosed with cancer or determined to still have cancer.

[0034] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human.

[0035] In some embodiments, the methods can further include generating a report, wherein the report comprises at least one of: (i) the cfDNA fragmentation profile based on the determined cfDNA fragment lengths; (ii) whether the subject has cancer; (iii) whether the level of cfDNA methylation in the subject is altered as compared to the level of cDNA methylation in a healthy subject; (iv) whether the methylation profile is characteristic of a methylation profile in a subject with cancer; (v) whether the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject: or (vi) whether the methylation profile is characteristic of a methylation profile in a subject with cancer.BRIEF DESCRIPTION OF FIGURES

[0036] Non-limiting and non-exhaustive examples are described with reference to the following figures.

[0037] FIG. 1 illustrates correlation plots comparing DELFI scores between different sequencing methods, according to aspects of the present disclosure.

[0038] FIG. 2 shows fragment width frequency distributions across multiple samples for different sequencing techniques, according to aspects of the present disclosure.71623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WO

[0039] FIG. 3 depicts cumulative distribution functions of fragment widths for TAPS, whole genome bisulfite sequencing (WGBS), and whole genome sequencing (WGS) methods, according to aspects of the present disclosure.

[0040] FIG. 4 illustrates scatter plots showing correlations between TAPS centered ratios across multiple samples, according to aspects of the present disclosure.

[0041] FIG. 5 shows scatter plots comparing WGS and WGBS centered ratio measurements across samples, according to aspects of the present disclosure.

[0042] FIG. 6 depicts correlation plots demonstrating aneuploidy detection capabilities using TAPS sequencing, according to aspects of the present disclosure.

[0043] FIG. 7 illustrates scatter plots showing WGBS performance limitations in aneuploidy detection, according to aspects of the present disclosure.DETAILED DESCRIPTION OF THE INVENTION

[0044] As used herein, the terms “fragmentation profile,” “position dependent differences in fragmentation patterns,” and “differences in fragment size and coverage in a position dependent manner across the genome” are equivalent and can be used interchangeably. In some cases, determining a cfDNA fragmentation profile in a mammal can be used for identifying a mammal as having cancer.

[0045] Described herein is the use of TAPS for methylation analysis to avoid degradation of input DNA that occurs with bisulfite treatment. This leaves fragmentomic signals intact, thus allowing detection of DNA fragments in the same sequencing reaction that provides methylation information. Whereas bisulfite sequencing requires two separate reactions. This simplifies the library preparation procedure, thus reducing hands-on time or robot time, as well as the potential for error. It also reduces the consumption of sequencing capacity, thus reducing costs.

[0046] Described herein is a method that allows the detection of the positioning of methylation signals in the context of specific cell-free DNA fragments. Such intra-fragment positional information could provide unique signals for the detection of cancer or other diseases. Because fragments are degraded by bisulfite treatment of DNA, such positioning information is lost in conventional bisulfite methylation sequencing.

[0047] In one aspect, the present disclosure provides a method of determining a cell free DNA (cfDNA) methylation profile and a cfDNA fragmentation profile in a single DNA sample from a subject including: (i) identifying 5-methylcytosine (5mC) in a target nucleic acid from the DNA sample including: a) modifying target nucleic acid in the DNA sample including: 1) adding a81623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WO blocking group to 5-hydroxymethylcytosine (5hmC) in the DNA sample; 2) converting the 5mC in the DNA sample to 5-carboxylcytosine (5caC) and / or 5-formylcytosine (5fC); and 3) converting the 5caC and / or 5fC to dihydrouracil (DHU) to provide a modified DNA sample including a modified target nucleic acid; b) preparing sequencing libraries; and c) generating a sequence of the modified target nucleic acid; wherein a cytosine (C) to thymine (T) transition in the sequence of the modified target nucleic acid compared to the target nucleic acid provides a location of a 5mC in the target nucleic acid; and (ii) subjecting the sequencing libraries obtained from step (i) to whole genome sequencing to obtain sequenced fragments; mapping the sequenced fragments to a genome to obtain genomic intervals of mapped sequences; and, analyzing the genomic intervals of mapped sequences to determine cfDNA fragment lengths and determining the cfDNA fragmentation profile using the lengths.

[0048] In one aspect, the present disclosure provides a method of determining a cell free DNA (cfDNA) methylation profile and a cfDNA fragmentation profile in a single DNA sample from a subject including: (i) identifying methylated cytosine in a target nucleic acid from the DNA sample including: a) modifying target nucleic acid in the DNA sample including performing enzymatic conversion of methylated cytosine; b) prepare sequencing libraries; and c) generating a sequence of the modified target nucleic acid; wherein a cytosine conversion in the sequence of the modified target nucleic acid compared to the target nucleic acid provides a location of a methyl cytosine in the target nucleic acid; and (ii) subjecting the sequencing libraries obtained from step (i) to whole genome sequencing to obtain sequenced fragments; mapping the sequenced fragments to a genome to obtain genomic intervals of mapped sequences; and, analyzing the genomic intervals of mapped sequences to determine cfDNA fragment lengths and determining the cfDNA fragmentation profile using the lengths.

[0049] In one aspect, the present disclosure provides a method of diagnosing cancer or screening for early detection of cancer in a subject including: (i) determining a cell free DNA (cfDNA) methylation profile including identifying 5-methylcytosine (5mC) in a target nucleic acid in a DNA sample from the subject including: a) modifying target nucleic acid in the sample including: 1) adding a blocking group to 5-hydroxymethylcytosine (5hmC) in the DNA sample; 2) converting the 5mC in the DNA sample to 5-carboxylcytosine (5caC) and / or 5-formylcytosine (5fC); and 3) converting the 5caC and / or 5fC to dihydrouracil (DHU) to provide a modified DNA sample including a modified target nucleic acid; b) preparing sequencing libraries; and c) generating a sequence of the modified target nucleic acid; wherein a cytosine (C) to thymine (T) transition in the sequence of the modified target nucleic acid compared to the target nucleic acid91623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WO provides a location of a 5mC in the target nucleic acid; (ii) determining a cfDNA fragmentation profile in the DNA sample from the subject including: a) subjecting the sequencing libraries obtained from step (i) to whole genome sequencing to obtain sequenced fragments; b) mapping the sequenced fragments to a genome to obtain genomic intervals of mapped sequences; and, c) analyzing the genomic intervals of mapped sequences to determine cfDNA fragment lengths and determining the cfDNA fragmentation profile using the lengths; and (ii) diagnosing the subject with cancer if the level of cfDNA methylation in the subject is altered as compared to the level of cDNA methylation in a healthy subject, the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject, or the level of cfDNA methylation in the subject is altered as compared to the level of cDNA methylation in a healthy subject and the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject.

[0050] In one aspect, the present disclosure provides a method of diagnosing cancer or screening for early detection of cancer in a subject comprising: (i) determining a cell free DNA (cfDNA) methylation profile comprising identifying 5-methylcytosine (5mC) in a target nucleic acid in a DNA sample from the subject comprising: a) modifying target nucleic acid in the sample comprising: 1) adding a blocking group to 5-hydroxymethylcytosine (5hmC) in the DNA sample; 2) converting the 5mC in the DNA sample to 5 -carboxylcytosine (5caC) and / or 5-formylcytosine (5fC); and 3) converting the 5caC and / or 5fC to dihydrouracil (DHU) to provide a modified DNA sample comprising a modified target nucleic acid; b) preparing sequencing libraries; and c) generating a sequence of the modified target nucleic acid; wherein a cytosine (C) to thymine (T) transition in the sequence of the modified target nucleic acid compared to the target nucleic acid provides a location of a 5mC in the target nucleic acid; (ii) determining a cfDNA fragmentation profile in the DNA sample from the subject comprising: a) subjecting the sequencing libraries obtained from step (i) to whole genome sequencing to obtain sequenced fragments; b) mapping the sequenced fragments to a genome to obtain genomic intervals of mapped sequences; and, c) analyzing the genomic intervals of mapped sequences to determine cfDNA fragment lengths and determining the cfDNA fragmentation profile using the lengths; and (iii) diagnosing the subject with cancer if the methylation profile is characteristic of a methylation profile in a subject with cancer, the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject, or the methylation profile is characteristic of a methylation profile in a subject with cancer and the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subj ect.101623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WO

[0051] In one aspect, the present disclosure provides a method of monitoring cancer treatment response in a subject in need thereof, including: (i) determining a cell free DNA (cfDNA) methylation profile including identifying 5-methylcytosine (5mC) in a target nucleic acid in a DNA sample from the subject including: a) modifying target nucleic acid in the DNA sample including: 1) adding a blocking group to 5 -hydroxy methylcytosine (5hmC) in the DNA sample; 2) converting the 5mC in the DNA sample to 5-carboxylcytosine (5caC) and / or 5-formylcytosine (5fC); and 3) converting the 5caC and / or 5fC to dihydrouracil (DHU) to provide a modified DNA sample including a modified target nucleic acid; b) preparing sequencing libraries; and c) generating a sequence of the modified target nucleic acid; wherein a cytosine (C) to thymine (T) transition in the sequence of the modified target nucleic acid compared to the target nucleic acid provides a location of a 5mC in the target nucleic acid; (ii) determining a cfDNA fragmentation profile in the DNA sample from the subject including: a) subjecting the sequencing libraries obtained from step (i) to whole genome sequencing to obtain sequenced fragments; b) mapping the sequenced fragments to a genome to obtain genomic intervals of mapped sequences; and c) analyzing the genomic interv als of mapped sequences to determine cfDNA fragment lengths and determining the cfDNA fragmentation profile using the lengths; and (iii) determining the subject still has cancer if the level of cfDNA methylation in the subject is altered as compared to the level of cDNA methylation in a healthy subject, the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject, or the level of cfDNA methylation in the subject is altered as compared to the level of cDNA methylation in a healthy subject and the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject.

[0052] In one aspect, the present disclosure provides a method of monitoring cancer treatment response in a subject in need thereof, including: (i) determining a cell free DNA (cfDNA) methylation profile including identifying 5-methylcytosine (5mC) in a target nucleic acid in a DNA sample from the subject including: a) modifying target nucleic acid in the DNA sample including: 1) adding a blocking group to 5-hydroxymethylcytosine (5hmC) in the DNA sample; 2) converting the 5mC in the DNA sample to 5-carboxylcytosine (5caC) and / or 5-formylcytosine (5fC); and 3) converting the 5caC and / or 5fC to dihydrouracil (DHU) to provide a modified DNA sample including a modified target nucleic acid; b) preparing sequencing libraries; and c) generating a sequence of the modified target nucleic acid; wherein a cytosine (C) to thymine (T) transition in the sequence of the modified target nucleic acid compared to the target nucleic acid provides a location of a 5mC in the target nucleic acid; (ii) determining a cfDNA fragmentation 111623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WO profile in the DNA sample from the subject including: a) subjecting the sequencing libraries obtained from step (i) to whole genome sequencing to obtain sequenced fragments; b) mapping the sequenced fragments to a genome to obtain genomic intervals of mapped sequences; and c) analyzing the genomic intervals of mapped sequences to determine cfDNA fragment lengths and determining the cfDNA fragmentation profile using the lengths; and (iii) determining the subject still has cancer if the methylation profile is characteristic of a methylation profile in a subject with cancer, the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject, or the methylation profile is characteristic of a methylation profile in a subject with cancer and the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject.

[0053] The present invention provides methods for identifying the location of methylated cytosine in a nucleic acid. The present invention provides methods for identifying the location of 5-methylcytosine in a nucleic acid. The methods described herein provide for DNA methylation analysis involving mild reactions that detect the modified cytosine quantitatively with baseresolution -without affecting the unmodified cytosine. Provided herein is a method for identifying 5mC by combining TET oxidation and reduction by borane derivatives (e.g., pyridine borane and 2-picoline borane (pic-BEE)), referred to herein as TAPS (TET-Assisted Pyridine borane Sequencing). TAPS detects modifications directly with high sensitivity and specificity, without affecting unmodified cytosines, and can be adopted to detect other cytosine modifications. It is non-destructive, preserving RNA and DNA up to 10 kbs long. Compared with bisulfite sequencing, TAPS results in higher mapping rates, more even coverage and lower sequencing costs, enabling higher quality, more comprehensive and cheaper methylome analyses. In some embodiments, identifying the methylated cytosine in a nucleic acid in a mammal can be used for identifying a mammal as having cancer. In some embodiments, identifying the location of 5- methylcytosine in a nucleic acid in a mammal can be used for identifying a mammal as having cancer. In some embodiments, the methylation analysis is as described in the International Patent Publication WO2019136413A1 (published Jul. 11, 2019), which is herein incorporated by reference in its entirety. Described herein are methods for further analyzing the sample used in the TAPS analysis to determine fragment profiles of cfDNA. In particular, the methylation analysis can include identifying the location of one or more of 5-methylcytosine, 5- hydroxymethylcytosine, 5-carboxylcytosine and / or 5 -formylcytosine in a target nucleic acid quantitatively with base-resolution without affecting the unmodified cytosine. In some embodiments, the target nucleic acid is DNA. In other embodiments, the target nucleic acid is121623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WORNA. Likewise, the nucleic acid sample that comprises the target nucleic acid may be a DNA sample or an RNA sample.

[0054] The target nucleic acid may be any nucleic acid having cytosine modifications (i. e. , 5mC, 5hmC, 5fC, and / or 5caC). The target nucleic acid can be a single nucleic acid molecule in the sample, or may be the entire population of nucleic acid molecules in a sample (or a subset thereof). The target nucleic acid can be the native nucleic acid from the source (e.g., cells or tissue samples) or can pre-converted into a high-throughput sequencing-ready form, for example by fragmentation, repair and ligation with adaptors for sequencing. Thus, target nucleic acids can comprise a plurality of nucleic acid sequences such that the methods described herein may be used to generate a library of target nucleic acid sequences that can be analyzed individually (e.g., by determining the sequence of individual targets) or in a group (e g., by high-throughput or next generation sequencing methods).

[0055] A nucleic acid sample can be obtained from an organism from the monera (bacteria), protista, fungi, plantae, and animalia kingdoms. Nucleic acid samples may be obtained from a from a patient or subject, from an environmental sample, or from an organism of interest. In embodiments, the nucleic acid sample is extracted or derived from a cell or collection of cells, a body fluid, a tissue sample, an organ, and an organelle.

[0056] The methods described herein can include identifying the location of one or more of 5- methylcytosine, 5- hydroxymethylcytosine, 5-carboxylcytosine and / or 5 -formylcytosine in a target RNA quantitatively with base-resolution without affecting the unmodified cytosine. In embodiments, the RNA is one or more of mRNA (messenger RNA). tRNA (transfer RNA). rRNA (ribosomal RNA), snRNA (small nuclear RNA), miRNA (microRNA), IncRNA (long noncoding RNA) and eRNA (enhancer RNA). The target RNA can be a single RNA molecule in the sample, or may be the entire population of RNA molecules in a sample (or a subset thereof). Thus, target RNA can comprise a plurality of RNA sequences such that the methods described herein may be used to generate a library of target RNA sequences that can be analyzed individually (e g., by determining the sequence of individual targets) or in a group (e.g., by high-throughput or next generation sequencing methods).

[0057] The methods described herein can utilize mild enzymatic and chemical reactions that avoid the substantial degradation associated with methods like bisulfite sequencing. Thus, the methods of the present invention are useful in analysis of low-input samples, such as circulating cell- free DNA and in single-cell analysis.131623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WO

[0058] A DNA sample used in the methods described herein may be from any source including, for example a body fluid, tissue sample, organ, organelle, or single cells. In embodiments, the DNA sample is circulating cell-free DNA (cell-free DNA or cfDNA). which is DNA found in the blood and is not present within a cell. cfDNA can be isolated from blood or plasma using methods known in the art. The DNA sample may result from an enrichment step, including, but is not limited to antibody immunoprecipitation, chromatin immunoprecipitation, restriction enzyme digestion-based enrichment, hybridization-based enrichment, or chemical labeling-based enrichment. The enrichment step may result in increased amounts of nucleosomal DNA, cfDNA, ctDNA or any combination thereof, compared to the amounts of nucleosomal DNA, cfDNA, ctDNA or any combination thereof, in the DNA sample prior to the enrichment step. In other words, the enrichment step results in an enriched DNA sample that differs from the DNA sample from any source including, for example a body fluid, tissue sample, organ, organelle, or single cells.

[0059] The target DNA may be any DNA having cytosine modifications (i.e., 5mC, 5hmC, 5fC, and / or 5caC) including, but not limited to, DNA fragments or genomic DNA purified from tissues, organs, cells and organelles. The target DNA can be a single DNA molecule in the sample, or maybe the entire population of DNA molecules in a sample (or a subset thereof). The target DNA can be the native DNA from the source or pre-converted into a high-throughput sequencing-ready form, for example by fragmentation, repair and ligation with adaptors for sequencing. Thus, target DNA can comprise a plurality of DNA sequences such that the methods described herein may be used to generate a library of target DNA sequences that can be analyzed individually (e.g., by determining the sequence of individual targets) or in a group (e g., by high-throughput or next generation sequencing methods).

[0060] In some embodiments, the TAPS method described herein can include the step of converting the 5mC and 5hmC (or just the 5mC if the 5hmC is blocked) to 5caC and / or 5fC. In embodiments of the invention, this step comprises contacting the DNA or RNA sample with a TET enzyme. The TET enzy mes are a family of enzy mes that catalyze the transfer of an oxygen molecule to the N5 methyl group on 5mC resulting in the formation of 5-hydroxymethylcytosine (5hmC). TET further catalyzes the oxidation of 5hmC to 5fC and the oxidation of 5fC to form 5caC. TET enzymes useful in the methods of the invention include one or more of human TET1, TET2, and TET3; murine Tetl, Tet2, and Tet3; Naegleria TET (NgTET); Coprinopsis cinerea (CcTET) and derivatives or analogues thereof. In embodiments, the TET enz me is NgTET. In other embodiments the TET enzyme is human TET1 (hTETl ).141623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WO

[0061] In some embodiments, the methods described herein can include the step of converting the 5caC and / or 5fC in a nucleic acid sample to DHU. In embodiments, this step comprises contacting the DNA or RNA sample with a reducing agent including, for example, a borane reducing agent such as pyridine borane, 2-picoline borane (pic-Bfb), borane, sodium borohydride, sodium cyanoborohydride, and sodium triacetoxyborohydride. In a preferred embodiment, the reducing agent is pyridine borane and / or pic-Bfb.

[0062] In some embodiments, the methods described herein can include the step of amplifying (increasing) the copy number of the modified target nucleic acid by a variety of different methods. When the modified target nucleic acid is DNA, the copy number can be increased by, for example, PCR, cloning, and primer extension. The copy number of individual target DNAs can be amplified by PCR using primers specific for a particular target DNA sequence. Alternatively, a plurality of different modified target DNA sequences can be amplified by cloning into a DNA vector by standard techniques. In embodiments of the invention, the copy number of a plurality of different modified target DNA sequences is increased by PCR to generate a library for next generation sequencing where, e.g., double-stranded adapter DNA has been previously ligated to the sample DNA (or to the modified sample DNA) and PCR is performed using primers complimentary to the adapter DNA.

[0063] In some embodiments, the method comprises the step of detecting the sequence of the modified target nucleic acid. The modified target DNA or RNA contains DHU at positions where one or more of 5mC. 5hmC, 5fC, and 5caC were present in the unmodified target DNA or RNA. DHU acts as a T in DNA replication and sequencing methods. Thus, the cytosine modifications can be detected by any direct or indirect method that identifies a C to T transition known in the art. Such methods include sequencing methods such as Sanger sequencing, microarray, and next generation sequencing methods. The C to T transition can also be detected by restriction enzyme analysis where the C to T transition abolishes or introduces a restriction endonuclease recognition sequence.

[0064] In some embodiments, determining a cfDNA fragmentation profile in a mammal can be used for identifying a mammal as having cancer. For example, cfDNA fragments obtained from a mammal (e.g., from a sample obtained from a mammal) can be subjected to low coverage wholegenome sequencing, and the sequenced fragments can be mapped to the genome (e.g., in nonoverlapping windows) and assessed to determine a cfDNA fragmentation profile. As described herein, a cfDNA fragmentation profile of a mammal having cancer is more heterogeneous (e.g., in fragment lengths) than a cfDNA fragmentation profile of a healthy mammal (e.g., a mammal 151623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WO not having cancer). As such, this document also provides methods and materials for assessing, monitoring, and / or treating mammals (e.g., humans) having, or suspected of having, cancer. In some embodiments, this document provides methods and materials for identifying a mammal as having cancer. For example, a sample (e g., a blood sample) obtained from a mammal can be assessed to determine the presence and, optionally, the tissue of origin of the cancer in the mammal based, at least in part, on the cfDNA fragmentation profile of the mammal. In some embodiments, this document provides methods and materials for monitoring a mammal as having cancer. For example, a sample (e.g., a blood sample) obtained from a mammal can be assessed to determine the presence of the cancer in the mammal based, at least in part, on the cfDNA fragmentation profile of the mammal. In some embodiments, this document provides methods and materials for identifying a mammal as having cancer, and administering one or more cancer treatments to the mammal to treat the mammal. For example, a sample (e.g., a blood sample) obtained from a mammal can be assessed to determine if the mammal has cancer based, at least in part, on the cfDNA fragmentation profile of the mammal, and one or more cancer treatments can be administered to the mammal.

[0065] In some embodiments, a fragmentation profile is determined using a fragmentation assay. Fragmentation assays can be performed at 10X to IX sequencing. In one embodiment, fragmentation assay is performed at 10X sequencing. In some embodiments, the fragmentation profile analysis is as described in the US Patent 10,982,279B2 (issued Apr. 20, 2021), which is herein incorporated by reference in its entirety. For example, cfDNA fragments obtained from a mammal (e.g.. from a sample obtained from a mammal) can be subjected to low coverage wholegenome sequencing, and the sequenced fragments can be mapped to the genome (e g., in nonoverlapping windows) and assessed to determine a cfDNA fragmentation profile. As described herein, a cfDNA fragmentation profile of a mammal having cancer is more heterogeneous (e.g., in fragment lengths) than a cfDNA fragmentation profile of a healthy mammal (e.g., a mammal not having cancer). As such, this document also provides methods and materials for assessing, monitoring, and / or treating mammals (e.g., humans) having, or suspected of having, cancer. In some cases, this document provides methods and materials for identifying a mammal as having cancer. For example, a sample (e.g., a blood sample) obtained from a mammal can be assessed to determine the presence and, optionally, the tissue of origin of the cancer in the mammal based, at least in part, on the cfDNA fragmentation profile of the mammal. In some cases, this document provides methods and materials for monitoring a mammal as having cancer. For example, a sample (e.g., a blood sample) obtained from a mammal can be assessed to determine the presence 161623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WO of the cancer in the mammal based, at least in part, on the cfDNA fragmentation profile of the mammal. In some cases, this document provides methods and materials for identifying a mammal as having cancer, and administering one or more cancer treatments to the mammal to treat the mammal. For example, a sample (e.g., a blood sample) obtained from a mammal can be assessed to determine if the mammal has cancer based, at least in part, on the cfDNA fragmentation profile of the mammal, and one or more cancer treatments can be administered to the mammal.

[0066] A cfDNA fragmentation profile can include one or more cfDNA fragmentation patterns. A cfDNA fragmentation pattern can include any appropriate cfDNA fragmentation pattern. Examples of cfDNA fragmentation patterns include, without limitation, median fragment size, fragment size distribution, ratio of small cfDNA fragments to large cfDNA fragments, and the coverage of cfDNA fragments. In some cases, a cfDNA fragmentation pattern includes two or more (e.g.. two, three, or four) of median fragment size, fragment size distribution, ratio of small cfDNA fragments to large cfDNA fragments, and the coverage of cfDNA fragments. In some cases, cfDNA fragmentation profile can be a genome-wide cfDNA profile (e.g., a genome-wide cfDNA profile in windows across the genome). In some cases, cfDNA fragmentation profile can be a targeted region profile. A targeted region can be any appropriate portion of the genome (e.g., a chromosomal region). Examples of chromosomal regions for which a cfDNA fragmentation profile can be determined as described herein include, without limitation, a portion of a chromosome (e.g., a portion of 2q, 4p, 5p, 6q, 7p, 8q, 9q, lOq, l lq, 12q, and / or 14q) and a chromosomal arm (e.g., a chromosomal arm of 8q, 13q, 1 Iq, and / or 3p). In some cases, a cfDNA fragmentation profile can include two or more targeted region profiles.

[0067] In some cases, a cfDNA fragmentation profile can be used to identify changes (e.g., alterations) in cfDNA fragment lengths. An alteration can be a genome-wide alteration or an alteration in one or more targeted regions / loci. A target region can be any region containing one or more cancer-specific alterations. Examples of cancer-specific alterations, and their chromosomal locations, include, without limitation, those shown in Table 3 (Appendix C) and those shown in Table 6 (Appendix F). In some cases, a cfDNA fragmentation profile can be used to identify (e.g., simultaneously identify) from about 10 alterations to about 500 alterations (e.g., from about 25 to about 500, from about 50 to about 500. from about 100 to about 500, from about 200 to about 500, from about 300 to about 500, from about 10 to about 400, from about 10 to about 300, from about 10 to about 200, from about 10 to about 100, from about 10 to about 50, from about 20 to about 400, from about 30 to about 300, from about 40 to about 200, from about171623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WO50 to about 100, from about 20 to about 100, from about 25 to about 75, from about 50 to about 250, or from about 100 to about 200, alterations).

[0068] In some cases, a cfDNA fragmentation profile can be used to detect tumor-derived DNA. For example, a cfDNA fragmentation profile can be used to detect tumor-derived DNA by comparing a cfDNA fragmentation profile of a mammal having, or suspected of having, cancer to a reference cfDNA fragmentation profile (e.g., a cfDNA fragmentation profile of a healthy mammal and / or a nucleosomal DNA fragmentation profile of healthy cells from the mammal having, or suspected of having, cancer). In some cases, a reference cfDNA fragmentation profile is a previously generated profile from a healthy mammal. For example, methods provided herein can be used to determine a reference cfDNA fragmentation profile in a healthy mammal, and that reference cfDNA fragmentation profile can be stored (e.g., in a computer or other electronic storage medium) for future comparison to a test cfDNA fragmentation profile in mammal having, or suspected of having, cancer. In some cases, a reference cfDNA fragmentation profile (e.g., a stored cfDNA fragmentation profile) of a healthy mammal is determined over the whole genome. In some cases, a reference cfDNA fragmentation profile (e.g., a stored cfDNA fragmentation profile) of a healthy mammal is determined over a subgenomic interval.

[0069] In some cases, a cfDNA fragmentation profile can be used to identify a mammal (e.g., a human) as having cancer (e.g., a colorectal cancer, a lung cancer, a breast cancer, a gastric cancer, a pancreatic cancer, a bile duct cancer, and / or an ovarian cancer).

[0070] A cfDNA fragmentation profile can include a cfDNA fragment size pattern. cfDNA fragments can be any appropriate size. For example, cfDNA fragment can be from about 50 base pairs (bp) to about 400 bp in length. As described herein, a mammal having cancer can have a cfDNA fragment size pattern that contains a shorter median cfDNA fragment size than the median cfDNA fragment size in a healthy mammal. A healthy mammal (e.g., a mammal not having cancer) can have cfDNA fragment sizes having a median cfDNA fragment size from about 166.6 bp to about 167.2 bp (e.g., about 166.9 bp). In some cases, a mammal having cancer can have cfDNA fragment sizes that are, on average, about 1.28 bp to about 2.49 bp (e.g., about 1.88 bp) shorter than cfDNA fragment sizes in a healthy mammal. For example, a mammal having cancer can have cfDNA fragment sizes having a median cfDNA fragment size of about 164.11 bp to about 165.92 bp (e.g., about 165.02 bp).

[0071] A cfDNA fragmentation profile can include a cfDNA fragment size distribution. As described herein, a mammal having cancer can have a cfDNA size distribution that is more variable than a cfDNA fragment size distribution in a healthy mammal. In some case, a size 181623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WO distribution can be within a targeted region. A healthy mammal (e.g., a mammal not having cancer) can have a targeted region cfDNA fragment size distribution of about 1 or less than aboutI. In some cases, a mammal having cancer can have a targeted region cfDNA fragment size distribution that is longer (e g., 10, 15, 20, 25, 30, 35, 40, 45, 50 or more bp longer, or any number of base pairs between these numbers) than a targeted region cfDNA fragment size distribution in a healthy mammal. In some cases, a mammal having cancer can have a targeted region cfDNA fragment size distribution that is shorter (e.g., 10, 15, 20, 25. 30. 35, 40, 45, 50 or more bp shorter, or any number of base pairs between these numbers) than a targeted region cfDNA fragment size distribution in a healthy mammal. In some cases, a mammal having cancer can have a targeted region cfDNA fragment size distribution that is about 47 bp smaller to about 30 bp longer than a targeted region cfDNA fragment size distribution in a healthy mammal. In some cases, a mammal having cancer can have a targeted region cfDNA fragment size distribution of. on average, a 10,I I, 12, 13, 14, 15, 15, 17, 18, 19, 20 or more bp difference in lengths of cfDNA fragments. For example, a mammal having cancer can have a targeted region cfDNA fragment size distribution of, on average, about a 13 bp difference in lengths of cfDNA fragments. In some case, a size distribution can be a genome-wide size distribution. A healthy mammal (e.g.. a mammal not having cancer) can have very similar distributions of short and long cfDNA fragments genomewide. In some cases, a mammal having cancer can have, genome-wide, one or more alterations (e.g., increases and decreases) in cfDNA fragment sizes. The one or more alterations can be any appropriate chromosomal region of the genome. For example, an alteration can be in a portion of a chromosome. Examples of portions of chromosomes that can contain one or more alterations in cfDNA fragment sizes include, without limitation, portions of 2q, 4p, 5p, 6q, 7p, 8q, 9q, lOq, 1 Iq, 12q, and 14q. For example, an alteration can be across a chromosome arm (e.g., an entire chromosome arm).

[0072] A cfDNA fragmentation profile can include a ratio of small cfDNA fragments to large cfDNA fragments and a correlation of fragment ratios to reference fragment ratios. As used herein, with respect to ratios of small cfDNA fragments to large cfDNA fragments, a small cfDNA fragment can be from about 100 bp in length to about 150 bp in length. As used herein, with respect to ratios of small cfDNA fragments to large cfDNA fragments, a large cfDNA fragment can be from about 151 bp in length to 220 bp in length. As described herein, a mammal having cancer can have a correlation of fragment ratios (e.g., a correlation of cfDNA fragment ratios to reference DNA fragment ratios such as DNA fragment ratios from one or more healthy mammals) that is lower (e.g., 2-fold lower, 3-fold lower, 4-fold lower, 5-fold lower, 6-fold lower, 7-fold191623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WO lower, 8-fold lower, 9-fold lower, 10-fold lower, or more) than in a healthy mammal. A healthy mammal (e.g., a mammal not having cancer) can have a correlation of fragment ratios (e.g., a correlation of cfDNA fragment ratios to reference DNA fragment ratios such as DNA fragment ratios from one or more healthy mammals) of about 1 (e g., about 0.96). In some cases, a mammal having cancer can have a correlation of fragment ratios (e.g., a correlation of cfDNA fragment ratios to reference DNA fragment ratios such as DNA fragment ratios from one or more healthy mammals) that is, on average, about 0. 19 to about 0.30 (e.g., about 0.25) lower than a correlation of fragment ratios (e.g., a correlation of cfDNA fragment ratios to reference DNA fragment ratios such as DNA fragment ratios from one or more healthy mammals) in a healthy mammal.

[0073] A cfDNA fragmentation profile can include coverage of all fragments. Coverage of all fragments can include windows (e.g., non-overlapping windows) of coverage. In some cases, coverage of all fragments can include windows of small fragments (e.g., fragments from about 100 bp to about 150 bp in length). In some cases, coverage of all fragments can include windows of large fragments (e.g., fragments from about 151 bp to about 220 bp in length).

[0074] In some cases, a cfDNA fragmentation profile can be used to identify the tissue of origin of a cancer (e.g., a colorectal cancer, a lung cancer, a breast cancer, a gastric cancer, a pancreatic cancer, a bile duct cancer, or an ovarian cancer). For example, a cfDNA fragmentation profile can be used to identify7a localized cancer. When a cfDNA fragmentation profile includes a targeted region profile, one or more alterations described herein (e.g., in Table 3 (Appendix C) and / or in Table 6 (Appendix F)) can be used to identify the tissue of origin of a cancer. In some cases, one or more alterations in chromosomal regions can be used to identify the tissue of origin of a cancer.

[0075] A cfDNA fragmentation profile can be obtained using any appropriate method. In some cases, cfDNA from a mammal (e.g., a mammal having, or suspected of having, cancer) can be processed into sequencing libraries which can be subjected to whole genome sequencing (e.g., low-coverage whole genome sequencing), mapped to the genome, and analyzed to determine cfDNA fragment lengths. Mapped sequences can be analyzed in non-overlapping windows covering the genome. Windows can be any appropriate size. For example, windows can be from thousands to millions of bases in length. As one non-limiting example, a window can be about 5 megabases (Mb) long. Any appropriate number of windows can be mapped. For example, tens to thousands of windows can be mapped in the genome. For example, hundreds to thousands of windows can be mapped in the genome. A cfDNA fragmentation profile can be determined within each window. In some cases, a cfDNA fragmentation profile can be obtained as described in Example 1. In some cases, a cfDNA fragmentation profile can be obtained as shown in FIG. 1.201623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WO

[0076] In some cases, methods and materials described herein also can include machine learning. For example, machine learning can be used for identifying an altered fragmentation profile (e.g., using coverage of cfDNA fragments, fragment size of cfDNA fragments, coverage of chromosomes, and mtDNA).

[0077] In some cases, methods and materials described herein can be the sole method used to identify a mammal (e.g., a human) as having cancer (e.g., a colorectal cancer, a lung cancer, a breast cancer, a gastric cancer, a pancreatic cancer, a bile duct cancer, and / or an ovarian cancer). For example, determining a cfDNA fragmentation profile can be the sole method used to identify a mammal as having cancer.

[0078] In some cases, methods and materials described herein can be used together with one or more additional methods used to identify a mammal (e.g., a human) as having cancer (e.g., a colorectal cancer, a lung cancer, a breast cancer, a gastric cancer, a pancreatic cancer, a bile duct cancer, and / or an ovarian cancer). Examples of methods used to identify a mammal as having cancer include, without limitation, identifying one or more cancer-specific sequence alterations, identifying one or more chromosomal alterations (e.g., aneuploidies and rearrangements), and identifying other cfDNA alterations. For example, determining a cfDNA fragmentation profile can be used together with identifying one or more cancer-specific mutations in a mammal's genome to identify a mammal as having cancer. For example, determining a cfDNA fragmentation profile can be used together with identifying one or more aneuploidies in a mammal's genome to identify a mammal as having cancer.

[0079] In some aspects, this document also provides methods and materials for assessing, monitoring, and / or treating mammals (e g., humans) having, or suspected of having, cancer. In some cases, this document provides methods and materials for identifying a mammal as having cancer. For example, a sample (e.g., a blood sample) obtained from a mammal can be assessed to determine if the mammal has cancer based, at least in part, on the cfDNA fragmentation profile of the mammal. In some cases, this document provides methods and materials for identifying the location (e.g., the anatomic site ortissue of origin) of a cancer in a mammal. For example, asample (e.g., a blood sample) obtained from a mammal can be assessed to determine the tissue of origin of the cancer in the mammal based, at least in part, on the cfDNA fragmentation profile of the mammal. In some cases, this document provides methods and materials for identifying a mammal as having cancer, and administering one or more cancer treatments to the mammal to treat the mammal. For example, a sample (e.g., a blood sample) obtained from a mammal can be assessed to determine if the mammal has cancer based, at least in part, on the cfDNA fragmentation profile211623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WO of the mammal, and administering one or more cancer treatments to the mammal. In some cases, this document provides methods and materials for treating a mammal having cancer. For example, one or more cancer treatments can be administered to a mammal identified as having cancer (e.g., based, at least in part, on the cfDNA fragmentation profile of the mammal) to treat the mammal. In some cases, during or after the course of a cancer treatment (e.g., any of the cancer treatments described herein), a mammal can undergo monitoring (or be selected for increased monitoring) and / or further diagnostic testing. In some cases, monitoring can include assessing mammals having, or suspected of having, cancer by, for example, assessing a sample (e.g., a blood sample) obtained from the mammal to determine the cfDNA fragmentation profile of the mammal as described herein, and changes in the cfDNA fragmentation profiles over time can be used to identify response to treatment and / or identify the mammal as having cancer (e.g., a residual cancer).

[0080] Any appropriate mammal can be assessed, monitored, and / or treated as described herein. A mammal can be a mammal having cancer. A mammal can be a mammal suspected of having cancer. Examples of mammals that can be assessed, monitored, and / or treated as described herein include, without limitation, humans, primates such as monkeys, dogs. cats, horses, cows, pigs, sheep, mice, and rats. For example, a human having, or suspected of having, cancer can be assessed to determine a cfDNA fragmentation profiled as described herein and, optionally, can be treated with one or more cancer treatments as described herein.

[0081] Any appropriate sample from a mammal can be assessed as described herein (e.g., assessed for a DNA fragmentation pattern). In some cases, a sample can include DNA (e.g., genomic DNA). In some cases, a sample can include cfDNA (e g., circulating tumor DNA (ctDNA)). In some cases, a sample can be enriched for nucleosomal DNA. In some cases, a sample can be enriched for ctDNA. In some cases, a sample can be enriched for cfDNA. In some cases, a sample can be fluid sample (e.g., a liquid biopsy). Examples of samples that can contain DNA and / or polypeptides include, without limitation, blood (e.g., whole blood, serum, or plasma), amnion, tissue, urine, cerebrospinal fluid, saliva, sputum, broncho-alveolar lavage, bile, lymphatic fluid, cyst fluid, stool, ascites, pap smears, breast milk, and exhaled breath condensate. For example, a plasma sample can be assessed to determine a cfDNA fragmentation profiled as described herein.

[0082] A sample from a mammal to be assessed as described herein (e.g., assessed for a DNA fragmentation pattern) can include any appropriate amount of cfDNA. In some cases, a sample can include a limited amount of DNA. For example, a cfDNA fragmentation profile can be 221623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WO obtained from a sample that includes less DNA than is typically required for other cfDNA analysis methods, such as those described in, for example, Phallen et al., 2017 Sci Transl Med 9; Cohen et al., 2018 Science 359:926; Newman et al., 2014 Nat Med 20:548; and Newman et al.. 2016 Nat Biotechnol 34:547).

[0083] In some cases, a sample can be processed (e.g., to isolate and / or purity- DNA and / or polypeptides from the sample). For example, DNA isolation and / or purification can include cell lysis (e.g., using detergents and / or surfactants), protein removal (e.g., using a protease), and / or RNA removal (e.g., using an RNase). As another example, polypeptide isolation and / or purification can include cell lysis (e.g., using detergents and / or surfactants), DNA removal (e.g., using a DNase), and / or RNA removal (e.g., using an RNase). In some cases, the sample can be processed in order to enrich for nucleosomal DNA, cfDNA. ctDNA, or any combination thereof.

[0084] A mammal having, or suspected of having, any appropriate ty pe of cancer can be assessed (e g., to determine a cfDNA fragmentation profile) and / or treated (e.g., by administering one or more cancer treatments to the mammal) using the methods and materials described herein. A cancer can be any stage cancer. In some cases, a cancer can be an early stage cancer. In some cases, a cancer can be an asymptomatic cancer. In some cases, a cancer can be a residual disease and / or a recurrence (e.g., after surgical resection and / or after cancer therapy). A cancer can be any ty pe of cancer. Examples of types of cancers that can be assessed, monitored, and / or treated as described herein include, without limitation, colorectal cancers, lung cancers, breast cancers, gastric cancers, pancreatic cancers, bile duct cancers, and ovarian cancers.

[0085] When treating a mammal having, or suspected of having, cancer as described herein, the mammal can be administered one or more cancer treatments. A cancer treatment can be any appropriate cancer treatment. One or more cancer treatments described herein can be administered to a mammal at any appropriate frequency (e.g., once or multiple times over a period of time ranging from days to weeks). Examples of cancer treatments include, without limitation adjuvant chemotherapy, neoadjuvant chemotherapy, radiation therapy, hormone therapy, cytotoxic therapy, immunotherapy, adoptive T cell therapy (e.g., chimeric antigen receptors and / or T cells having wild-type or modified T cell receptors), targeted therapy such as administration of kinase inhibitors (e.g., kinase inhibitors that target a particular genetic lesion, such as a translocation or mutation), (e.g. a kinase inhibitor, an antibody, a bispecific antibody), signal transduction inhibitors, bispecific antibodies or antibody fragments (e.g., BiTEs), monoclonal antibodies, immune checkpoint inhibitors, surgery- (e.g., surgical resection), or any combination of the above.231623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WOIn some cases, a cancer treatment can reduce the severity of the cancer, reduce a symptom of the cancer, and / or to reduce the number of cancer cells present within the mammal.

[0086] In some embodiments, the methods described herein can further include generating a report embodying the results of the cfDNA methylation profile and / or the cfDNA fragmentation profile, as determined using the techniques described above. In some embodiments, the report can include the cfDNA fragmentation profile based on the determined cfDNA fragment lengths. In some embodiments, the report can include whether the subject has cancer. In some embodiments, the report can include whether the level of cfDNA methylation in the subject is altered as compared to the level of cDNA methylation in a healthy subject. In some embodiments, the report can include whether the methylation profile is characteristic of a methylation profile in a subject with cancer. In some embodiments, the report can include whether the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject. In some embodiments, the report can include whether the level of cfDNA methylation in the subject is altered as compared to the level of cDNA methylation in a healthy subject and the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject. In some embodiments, the report can include whether the methylation profile is characteristic of a methylation profile in a subject with cancer and the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject. In some embodiments, the report or reports can include any combination of the information described above. In any of these embodiments, the generated reports can be useful for allowing medical practitioners to monitor cancer progression in a subject and / or informing cancer treatment decisions (e.g., facilitating the ability of medical practitioners to select appropriate cancer treatments given the progression and / or status of the subject’s cancer condition).

[0087] In some embodiments, the DNA sample comprises picogram quantities of DNA. In some embodiments, the DNA sample comprises about 1 pg to about 900 pg DNA. about 1 pg to about 500 pg DNA, about 1 pg to about 100 pg DNA, about 1 pg to about 50 pg DNA, about 1 to about 10 pg, DNA, less than about 200 pg, less than about 100 pg DNA, less than about 50 pg DNA, less than about 20 pg DNA, and less than about 5 pg DNA. In other embodiments, the DNA sample comprises nanogram quantities of DNA. In some embodiments, the DNA sample contains about 1 to about 500 ng of DNA, about 1 to about 200 ng of DNA, about 1 to about 100 ng of DNA, about 1 to about 50 ng of DNA, about 1 ng to about 10 ng of DNA, about 1 ng to about 5 ng of DNA, less than about 100 ng of DNA, less than about 50 ng of DNA less than about 5 ng of DNA, or less that about 2 ng of DNA. In some embodiments, the DNA sample comprises 241623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WO circulating cell-free DNA (cfDNA). In embodiments of the invention the DNA sample comprises microgram quantities of DNA.

[0088] In some embodiments, the step of generating the sequence of the modified target nucleic acid comprises one or more of chain termination sequencing, microarray, high-throughput sequencing, and restriction enzyme analysis.

[0089] In some embodiments, the percentages of a T at each transition location provide a quantitative level of 5mC at each location in the target nucleic acid.

[0090] In some embodiments, the mapped sequences comprise tens or hundreds to thousands of genomic intervals. In some embodiments, the genomic intervals are non-overlapping. In some embodiments, the genomic intervals each comprise thousands to millions of base pairs. In some embodiments, a cfDNA fragmentation profile is determined within each genomic intervals.

[0091] In some embodiments, the cfDNA fragmentation profile comprises a median fragment size. In some embodiments, the cfDNA fragmentation profile comprises a fragment size distribution. In some embodiments, the cfDNA fragmentation profile comprises a ratio of small cfDNA fragments to large cfDNA fragments in said windows of mapped sequences. In some embodiments, the cfDNA fragmentation profile comprises the sequence coverage of small cfDNA fragments in genomic intervals across the genome. In some embodiments, the cfDNA fragmentation profile comprises the sequence coverage of large cfDNA fragments in genomic intervals across the genome. In some embodiments, the cfDNA fragmentation profile comprises the sequence coverage of small and large cfDNA fragments in genomic intervals across the genome.

[0092] In some embodiments, methylation profiles characteristic of cancer are determined using prior machine learning in a subject with cancer compared to methylation profiles in a healthy subj ect.

[0093] In some embodiments, methylation profiles characteristic of cancer are determined based on methylation profiles described in the scientific literature.

[0094] In some embodiments, methylation profiles characteristic cancer are determined based on a combination of using prior machine learning in a subject with cancer compared to methylation profiles in a healthy subject and methylation profiles described in the scientific literature.

[0095] In some embodiments, the methylation profiles characteristic of cancer are determined by increased level of cfDNA methylation in the subject as compared to level of cfDNA methylation in a healthy subject.251623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WO

[0096] In some embodiments, the methylation profiles characteristic of cancer incorporate the position of methylated cytosine with respect to the ends of cfDNA fragments.

[0097] In some embodiments, genome coverage is about 9x to 0. lx.

[0098] In some embodiments, the cancer is selected from the group consisting of: colorectal cancer, lung cancer, breast cancer, gastric cancer, pancreatic cancer, bile duct cancer, and ovarian cancer.

[0099] In some embodiments, a therapeutic treatment suitable for treatment of the cancer is administered to the subject if the subject is diagnosed with cancer or determined to still have cancer.

[0100] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human.

[0101] FIGs. 1-7 demonstrate experimental results comparing the performance of TAPS using the techniques described herein for recovering both fragmentation and methylation information against WGS, which is used for recovering fragmentation information, and WGBS, which is used for recovering methylation information. The enzymatic conversion of methylated cytosine may be performed using TAPS as a specific implementation of the general enzymatic conversion method described herein. In some cases, the blocking group added to 5-hydroxymethylcytosine may be specifically implemented using pyridine borane or 2-picoline borane (pic-BEh) as the borane derivative. The experimental comparison utilized DNA samples comprising nanogram quantities of DNA. specifically about 10 ng of cfDNA for TAPS library preparation and about 1 ng of cfDNA for DELFI fragmentation assays. The fragmentation assays were performed at 10X sequencing coverage, demonstrating that sequencing coverage ranging from 10X to IX sequencing may be utilized for such analyses.

[0102] Referring to FIG. 1, TAPS produces similar DELFI scores to WGS data, as demonstrated by the correlation analysis shown in the scatter plot with linear regression line and the comparative box plots. DELFI scores can be calculated based on a cfDNA fragmentation profile. In some aspects, calculating the DELFI score includes: i) determining a ratio of short to long cfDNA fragments of the sample, ii) determining a Z-score for cfDNA fragments of the sample by chromosome arm, iii) quantifying cfDNA fragment density using a computational mixture model analysis, and iv) using a machine learning model to process output of i)-iii) to define the DELFI score. Additional information on the DELFI scores can be found in PCT Application Publication No. WO2022216981 Al, titled METHOD OF DETECTING CANCER USING GENOME- WIDE CFDNA FRAGMENTATION PROFILES, filed April 7, 2022. which is hereby incorporated by 261623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WO reference herein in its entirety. In various aspects, the score is utilized to determine a likelihood of overall survival of the subject. The data points show a strong linear relationship between the DELFI WGS scores and the corresponding TAPS-derived scores, with confidence intervals indicating statistical reliability of the correlation. This demonstrates that the enzymatic conversion approach maintains the fragmentation signal integrity compared to standard WGS approaches. The method preserves RNA and DNA up to 10 kbs long due to the non-destructive nature of the enzymatic conversion compared to bisulfite treatment, which contributes to the maintained signal quality observed in the correlation analysis.

[0103] As shown in FIG. 2, TAPS can reproduce fragment length distributions while WGBS cannot maintain such distributions. The 16 sample plots display three overlaid frequency distribution lines representing TAPS, WGBS, and WGS methodologies across different sample identifiers. The TAPS frequency distributions closely match the WGS distributions, with peaks occurring around the 150-200 base pair width range, while WGBS frequency distributions show altered patterns that do not correspond to the expected fragment length profiles. In some cases, the method may detect positioning of methylation signals in the context of specific cell-free DNA fragments, providing intra-fragment positional information for disease detection signals that would be lost with bisulfite treatment approaches. The preservation of fragment length information enables simultaneous analysis of both methylation patterns and fragmentation characteristics from the same DNA sample.

[0104] With continued reference to the experimental data, FIG. 3 illustrates that TAPS has consistent fragment width shifts of approximately 2 base pairs while WGBS shows inconsistent patterns in cumulative distribution function analysis. The 16 cumulative distribution plots demonstrate that the rightw ard shift in TAPS data remains consistent across all replicates, whereas the shift in WGBS data varies inconsistently and the overall fragment length distribution is not maintained. The green curves representing WGBS show steeper rises and reach higher cumulative values more rapidly than the blue and purple curves representing WGS and TAPS respectively. This consistency in TAPS performance indicates that the method results in higher mapping rates, more even coverage and lower sequencing costs compared to bisulfite sequencing methods, while maintaining reproducible fragmentation signal characteristics.

[0105] As demonstrated in FIG. 4, TAPS centered ratios show high correlation with WGS centered ratios across multiple samples. The 16 scatter plots, each labeled with unique sample identifiers, display clear positive linear correlations between WGS centered ratio values and TAPS centered ratio values, with red trend lines fitting closely to the scattered data points. The 271623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WO correlation strength remains consistent across different samples, indicating that TAPS maintains the quantitative relationships observed in standard WGS fragmentation analysis. In contrast, FIG. 5 shows that WGBS cannot reproduce the centered ratios demonstrated by TAPS, with scatter plots showing poor correlations and inconsistent relationships between WGS and WGBS centered ratio measurements across the same sample set.

[0106] FIG. 6 demonstrates that TAPS can capture aneuploidy detection capabilities equivalent to WGS analysis. The grid of 39 scatter plots labeled zOl through z39 shows correlation analyses with data points clustered along diagonal patterns in most cases, indicating that TAPS maintains the ability to detect chromosomal copy number variations. Each scatter plot displays varying degrees of linear relationships with trend lines, demonstrating that the enzymatic conversion approach does not compromise the detection of genomic alterations associated with cancer. The method may be applied to detect other cytosine modifications beyond 5-methylcytosine while maintaining these aneuploidy detection capabilities, and may be used for identifying tissue of origin of cancer in addition to cancer diagnosis through the combined analysis of methylation and fragmentation patterns.

[0107] In contrast to the TAPS performance shown in FIG. 6, FIG. 7 illustrates the limitations of WGBS in capturing aneuploidy signals. The collection of 31 scatter plots shows inconsistent correlations and reduced signal qualify compared to the TAPS analysis, with WGBS being subject to GC bias that affects coverage in high GC content regions. The data points show vary ing degrees of correlation along trend lines, with some plots displaying poor clustering around the regression lines. This demonstrates that bisulfite treatment compromises the ability to detect chromosomal alterations due to the harsh chemical conditions that degrade DNA. The method may be applied to diseases other than cancer, including infectious disease and autoimmune disorders, where both methylation and fragmentation analysis provide diagnostic value. Additionally, the method may analyze single-cell sequencing samples due to the preservation of low-input DNA samples, with DNA samples comprising picogram quantities ranging from about 1 pg to about 900 pg DNA, about 1 pg to about 500 pg DNA, about 1 pg to about 100 pg DNA, about 1 pg to about 50 pg DNA, or about 1 to about 10 pg DNA being suitable for analysis.

[0108] The experimental results demonstrate that TAPS maintains signal from cfDNA fragmentation assays across multiple fragmentation features while WGBS cannot maintain such signals. The combined analysis approach enables simultaneous profiling of methylation and fragmentation characteristics from a single DNA sample, providing advantages over separate analytical workflows that would require larger sample inputs and increased processing 281623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WO complexity. DNA samples comprising microgram quantities may also be utilized as alternative input amounts for applications requiring higher DNA concentrations, demonstrating the flexibility of the enzymatic conversion approach across different sample types and concentrations.

[0109] Although the invention has been described with reference to the presently preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

[0110] While various illustrative embodiments incorporating the principles of the present teachings have been disclosed, the present teachings are not limited to the disclosed embodiments. Instead, this application is intended to cover any variations, uses, or adaptations of the present teachings and use its general principles. Further, this application is intended to cover such departures from the present disclosure as come within known or customary’ practice in the art to which these teachings pertain.[OHl] In the above detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless con-text dictates otherwise. The illustrative embodiments described in the present disclosure are not meant to be limiting. Other embodiments may be used, and other changes may’ be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that various features of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.

[0112] The present disclosure is not to be limited in terms of the particular embodiments described in this application, which are intended as illustrations of various features. Many modifications and variations can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the dis-closure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. It is to be understood that this disclosure is not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0113] With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity.291623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WO

[0114] It will be understood by those within the art that, in general, terms used herein are generally in-tended as “open” terms (for example, the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” et cetera). While various compositions, methods, and devices are described in terms of “comprising” various components or steps (interpreted as meaning “including, but not limited to”), the compositions, methods, and devices can also “consist essentially of or “consist of the various components and steps, and such terminology should be interpreted as defining essentially closed-member groups.

[0115] In addition, even if a specific number is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (for example, the bare recitation of "two recitations," without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, et cetera” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (for example, “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together. A and C together, B and C together, and / or A, B, and C together, et cetera). In those instances where a convention analogous to “at least one of A, B, or C, et cetera” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (for example, “a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together. A and C together, B and C together, and / or A, B. and C together, et cetera). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting tw o or more alternative terms, whether in the description, sample embodiments, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” w ill be understood to include the possibilities of “A” or “B” or “A and B.”

[0116] In addition, where features of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0117] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, 301623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WO thirds, quarters, fifths, tenths, et cetera. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, et cetera. As will also be understood by one skilled in the art. all language such as ‘‘up to,” '"at least,” and the like include the number recited and refer to ranges that can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3. 4, or 5 cells, and so forth.

[0118] The term ‘‘about,” as used herein, refers to variations in a numerical quantity that can occur, for example, through measuring or handling procedures in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of compositions or reagents; and the like. Typically, the term “about” as used herein means greater or lesser than the value or range of values stated by 1 / 10 of the stated values, e.g., ±10%. The term “about” also refers to variations that would be recognized by one skilled in the art as being equivalent so long as such variations do not encompass known values practiced by the prior art. Each value or range of values preceded by the term “about” is also intended to encompass the embodiment of the stated absolute value or range of values. Whether or not modified by the term “about,” quantitative values recited in the present disclosure include equivalents to the recited values, e.g., variations in the numerical quantity of such values that can occur, but would be recognized to be equivalents by a person skilled in the art.

[0119] Various of the above-disclosed and other features and functions, or alternatives thereof, may be combined into many other different systems or applications. Various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art, each of which is also intended to be encompassed by the dis-closed embodiments.

[0120] The functions and process steps herein may be performed automatically or wholly or partially in response to user command. An activity (including a step) performed automatically is performed in response to one or more executable instructions or device operation without user direct initiation of the activity.311623885965.1

Claims

PATENTATTORNEY DOCKET NO. DELFI2170-1WOWhat Is Claimed Is:E A method of determining a cell free DNA (cfDNA) methylation profile and a cfDNA fragmentation profile in a single DNA sample from a subject comprising:(i) identifying 5 -methylcytosine (5mC) in a target nucleic acid from the DNA sample comprising: a) modifying target nucleic acid in the DNA sample comprising:1) adding a blocking group to 5-hydroxymethylcytosine (5hmC) in the DNA sample;2) converting the 5mC in the DNA sample to 5-carboxylcytosine (5caC) and / or 5-formylcytosine (5fC); and3) converting the 5caC and / or 5fC to dihydrouracil (DHU) to provide a modified DNA sample comprising a modified target nucleic acid; b) preparing sequencing libraries; and c) generating a sequence of the modified target nucleic acid; wherein a cytosine (C) to thymine (T) transition in the sequence of the modified target nucleic acid compared to the target nucleic acid provides a location of a 5mC in the target nucleic acid; and(ii) subjecting the sequencing libraries obtained from step (i) to whole genome sequencing to obtain sequenced fragments; mapping the sequenced fragments to a genome to obtain genomic intervals of mapped sequences; and, analyzing the genomic intervals of mapped sequences to determine cfDNA fragment lengths and determining the cfDNA fragmentation profile using the lengths.

2. A method of determining a cell free DNA (cfDNA) methylation profile and a cfDNA fragmentation profile in a single DNA sample from a subject comprising:(i) identifying methylated cytosine in a target nucleic acid from the DNA sample comprising: a) modifying target nucleic acid in the DNA sample comprising performing enzymatic conversion of methylated cytosine; b) prepare sequencing libraries; and c) generating a sequence of the modified target nucleic acid;321623885965.1PATENTATTORNEY DOCKET NO. DELFI2170-1WO wherein a cytosine conversion in the sequence of the modified target nucleic acid compared to the target nucleic acid provides a location of a methyl cytosine in the target nucleic acid; and(ii) subjecting the sequencing libraries obtained from step (i) to whole genome sequencing to obtain sequenced fragments; mapping the sequenced fragments to a genome to obtain genomic intervals of mapped sequences; and, analyzing the genomic intervals of mapped sequences to determine cfDNA fragment lengths and determining the cfDNA fragmentation profile using the lengths.

3. A method of diagnosing cancer or screening for early detection of cancer in a subject comprising:(i) determining a cell free DNA (cfDNA) methylation profile comprising identifying 5- methylcytosine (5mC) in a target nucleic acid in a DNA sample from the subject comprising: a) modifying target nucleic acid in the sample comprising:1) adding a blocking group to 5-hydroxymethylcytosine (5hmC) in the DNA sample;2) converting the 5mC in the DNA sample to 5-carboxylcytosine (5caC) and / or 5-formylcytosine (5fC); and3) converting the 5caC and / or 5fC to dihydrouracil (DHU) to provide a modified DNA sample comprising a modified target nucleic acid; b) preparing sequencing libraries; and c) generating a sequence of the modified target nucleic acid; wherein a cytosine (C) to thymine (T) transition in the sequence of the modified target nucleic acid compared to the target nucleic acid provides a location of a 5mC in the target nucleic acid;(ii) determining a cfDNA fragmentation profile in the DNA sample from the subject comprising: a) subjecting the sequencing libraries obtained from step (i) to whole genome sequencing to obtain sequenced fragments; b) mapping the sequenced fragments to a genome to obtain genomic intervals of mapped sequences; and,331623885965.1PATENTATTORNEY DOCKET NO. DELFI2170-1WO c) analyzing the genomic intervals of mapped sequences to determine cfDNA fragment lengths and determining the cfDNA fragmentation profile using the lengths; and diagnosing the subject with cancer if the level of cfDNA methylation in the subject is altered as compared to the level of cDNA methylation in a healthy subject, the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject, or the level of cfDNA methylation in the subject is altered as compared to the level of cDNA methylation in a healthy subject and the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject.

4. A method of diagnosing cancer or screening for early detection of cancer in a subject comprising:(iii) determining a cell free DNA (cfDNA) methylation profile comprising identifying 5- methylcytosine (5mC) in a target nucleic acid in a DNA sample from the subject comprising: a) modifying target nucleic acid in the sample comprising:1) adding a blocking group to 5-hydroxymethylcytosine (5hmC) in the DNA sample;2) converting the 5mC in the DNA sample to 5-carboxylcytosine (5caC) and / or 5-formylcytosine (5fC); and3) converting the 5caC and / or 5fC to dihydrouracil (DHU) to provide a modified DNA sample comprising a modified target nucleic acid; b) preparing sequencing libraries; and c) generating a sequence of the modified target nucleic acid; wherein a cytosine (C) to thymine (T) transition in the sequence of the modified target nucleic acid compared to the target nucleic acid provides a location of a 5mC in the target nucleic acid;(iv) determining a cfDNA fragmentation profile in the DNA sample from the subject comprising: a) subjecting the sequencing libraries obtained from step (i) to whole genome sequencing to obtain sequenced fragments;341623885965.1PATENTATTORNEY DOCKET NO. DELFI2170-1WO b) mapping the sequenced fragments to a genome to obtain genomic intervals of mapped sequences; and, c) analyzing the genomic intervals of mapped sequences to determine cfDNA fragment lengths and determining the cfDNA fragmentation profile using the lengths; and(v) diagnosing the subject with cancer if the methylation profile is characteristic of a methylation profile in a subject with cancer, the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject, or the methylation profile is characteristic of a methylation profile in a subject with cancer and the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject.

5. A method of monitoring cancer treatment response in a subject in need thereof, comprising:(i) determining a cell free DNA (cfDNA) methylation profile comprising identifying 5- methylcytosine (5mC) in a target nucleic acid in a DNA sample from the subject comprising: a) modifying target nucleic acid in the DNA sample comprising:1) adding a blocking group to 5-hydroxymethylcytosine (5hmC) in the DNA sample;2) converting the 5mC in the DNA sample to 5-carboxylcytosine (5caC) and / or 5-formylcytosine (5fC); and3) converting the 5caC and / or 5fC to dihydrouracil (DHU) to provide a modified DNA sample comprising a modified target nucleic acid; b) preparing sequencing libraries; and c) generating a sequence of the modified target nucleic acid; wherein a cytosine (C) to thymine (T) transition in the sequence of the modified target nucleic acid compared to the target nucleic acid provides a location of a 5mC in the target nucleic acid;(ii) determining a cfDNA fragmentation profile in the DNA sample from the subject comprising: a) subjecting the sequencing libraries obtained from step (i) to whole genome sequencing to obtain sequenced fragments;351623885965.1PATENTATTORNEY DOCKET NO. DELFI2170-1WO b) mapping the sequenced fragments to a genome to obtain genomic intervals of mapped sequences; and, c) analyzing the genomic intervals of mapped sequences to determine cfDNA fragment lengths and determining the cfDNA fragmentation profile using the lengths; and(iii) determining the subject still has cancer if the level of cfDNA methylation in the subject is altered as compared to the level of cDNA methylation in a healthy subject, the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject, or the level of cfDNA methylation in the subject is altered as compared to the level of cDNA methylation in a healthy subject and the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject.

6. A method of monitoring cancer treatment response in a subject in need thereof, comprising:(i) determining a cell free DNA (cfDNA) methylation profile comprising identifying 5- methylcytosine (5mC) in a target nucleic acid in a DNA sample from the subject comprising: a) modifying target nucleic acid in the DNA sample comprising:1) adding a blocking group to 5 -hydroxy methylcy tosine (5hmC) in the DNA sample;2) converting the 5mC in the DNA sample to 5-carboxylcytosine (5caC) and / or 5-formylcytosine (5fC); and3) converting the 5caC and / or 5fC to dihydrouracil (DHU) to provide a modified DNA sample comprising a modified target nucleic acid; b) preparing sequencing libraries; and c) generating a sequence of the modified target nucleic acid; wherein a cytosine (C) to thymine (T) transition in the sequence of the modified target nucleic acid compared to the target nucleic acid provides a location of a 5mC in the target nucleic acid;(ii) determining a cfDNA fragmentation profile in the DNA sample from the subject comprising: a) subjecting the sequencing libraries obtained from step (i) to whole genome sequencing to obtain sequenced fragments;361623885965.1PATENTATTORNEY DOCKET NO. DELFI2170-1WO b) mapping the sequenced fragments to a genome to obtain genomic intervals of mapped sequences; and, c) analyzing the genomic intervals of mapped sequences to determine cfDNA fragment lengths and determining the cfDNA fragmentation profile using the lengths; and(iii) determining the subject still has cancer if the methylation profile is characteristic of a methylation profile in a subject with cancer, the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject, or the methylation profile is characteristic of a methylation profile in a subject with cancer and the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject.

7. A method of treating cancer in a subject in need thereof, comprising:(i) determining a cell free DNA (cfDNA) methylation profile comprising identifying 5- methylcytosine (5mC) in a target nucleic acid in a DNA sample from the subject comprising: a) modifying target nucleic acid in the DNA sample comprising:1) adding a blocking group to 5-hydroxymethylcytosine (5hmC) in the DNA sample;2) converting the 5mC in the DNA sample to 5-carboxylcytosine (5caC) and / or 5-formylcytosine (5fC); and3) converting the 5caC and / or 5fC to dihydrouracil (DHU) to provide a modified DNA sample comprising a modified target nucleic acid; b) preparing sequencing libraries; and c) generating a sequence of the modified target nucleic acid. wherein a cytosine (C) to thymine (T) transition in the sequence of the modified target nucleic acid compared to the target nucleic acid provides a location of a 5mC in the target nucleic acid;(ii) determining a cfDNA fragmentation profile in the DNA sample from the subject comprising: a) subjecting the sequencing libraries obtained from step (i) to whole genome sequencing to obtain sequenced fragments;371623885965.1PATENT ATTORNEY DOCKET NO. DELFI2170-1WO b) mapping the sequenced fragments to a genome to obtain genomic intervals of mapped sequences; and, c) analyzing the genomic intervals of mapped sequences to determine cfDNA fragment lengths and determining the cfDNA fragmentation profile using the lengths;(iii) determining the subject still has cancer if the level of cfDNA methylation in the subject is altered as compared to the level of cDNA methylation in a healthy subject, the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject, or the level of cfDNA methylation in the subject is altered as compared to the level of cDNA methylation in a healthy subject and the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject; and(iv) in response to determining the subject still has cancer, treating the subject with a cancer treatment consistent with the cancer.

8. A method of treating cancer in a subject in need thereof, comprising:(i) determining a cell free DNA (cfDNA) methylation profile comprising identifying 5- methylcytosine (5mC) in a target nucleic acid in a DNA sample from the subject comprising: a) modifying target nucleic acid in the DNA sample comprising:1) adding a blocking group to 5-hydroxymethylcytosine (5hmC) in the DNA sample;2) converting the 5mC in the DNA sample to 5-carboxylcytosine (5caC) and / or 5-formylcytosine (5fC); and3) converting the 5caC and / or 5fC to dihydrouracil (DHU) to provide a modified DNA sample comprising a modified target nucleic acid; b) preparing sequencing libraries; and c) generating a sequence of the modified target nucleic acid. wherein a cytosine (C) to thymine (T) transition in the sequence of the modified target nucleic acid compared to the target nucleic acid provides a location of a 5mC in the target nucleic acid;(ii) determining a cfDNA fragmentation profile in the DNA sample from the subject compnsmg:381623885965.1PATENTATTORNEY DOCKET NO. DELFI2170-1WO a) subjecting the sequencing libraries obtained from step (i) to whole genome sequencing to obtain sequenced fragments; b) mapping the sequenced fragments to a genome to obtain genomic intervals of mapped sequences; and, c) analyzing the genomic intervals of mapped sequences to determine cfDNA fragment lengths and determining the cfDNA fragmentation profile using the lengths;(iii) determining the subject still has cancer if the methylation profile is characteristic of a methylation profile in a subject with cancer, the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject, or the methylation profile is characteristic of a methylation profile in a subject with cancer and the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject; and(iv) in response to determining the subject still has cancer, treating the subject with a cancer treatment consistent with the cancer.

9. The method of claims 1-8, wherein the step of generating the sequence of the modified target nucleic acid comprises one or more of chain termination sequencing, microarray, high- throughput sequencing, and restriction enzyme analysis.

10. The method of claims 1-9. wherein the percentages of a T at each transition location provide a quantitative level of 5mC at each location in the target nucleic acid.

11. The method of claims 1-9, wherein the mapped sequences comprise tens or hundreds to thousands of genomic intervals.

12. The method of claims 1-9, wherein the genomic intervals are non-overlapping.

13. The method of claims 1-9. wherein the genomic intervals each comprise thousands to millions of base pairs.

14. The method of claims 1-9, wherein a cfDNA fragmentation profile is determined w ithin each genomic intervals.391623885965.1PATENTATTORNEY DOCKET NO. DELFI2170-1WO15. The method of claims 1-9, wherein the cfDNA fragmentation profile comprises a median fragment size.

16. The method of claims 1-9, wherein the cfDNA fragmentation profile comprises a fragment size distribution.

17. The method of claims 1-9, wherein the cfDNA fragmentation profile comprises a ratio of small cfDNA fragments to large cfDNA fragments in said windows of mapped sequences.

18. The method of claims 1-9, wherein the cfDNA fragmentation profile comprises the sequence coverage of small cfDNA fragments in genomic intervals across the genome.

19. The method of claims 1-9, wherein the cfDNA fragmentation profile comprises the sequence coverage of large cfDNA fragments in genomic intervals across the genome.

20. The method of claims 1-9, wherein the cfDNA fragmentation profile comprises the sequence coverage of small and large cfDNA fragments in genomic intervals across the genome.

21. The method of claims 3-20, wherein the cancer is selected from the group consisting of: colorectal cancer, lung cancer, breast cancer, gastric cancer, pancreatic cancer, bile duct cancer, and ovarian cancer.

22. The method of claims 3-21, wherein methylation profiles characteristic of cancer are determined using prior machine learning in a subject with cancer compared to methylation profiles in a healthy subject.

23. The method of claims 3-21, wherein methylation profiles characteristic of cancer are determined based on methylation profiles described in the scientific literature.

24. The method of claims 3-21, wherein methylation profiles characteristic cancer are determined based on a combination of using prior machine learning in a subject with cancer compared to401623885965.1PATENTATTORNEY DOCKET NO. DELFI2170-1WO methylation profiles in a healthy subject and methylation profiles described in the scientific literature.

25. The method of claims 3-24, wherein the methylation profiles characteristic of cancer are determined by increased level of cfDNA methylation in the subject as compared to level of cfDNA methylation in a healthy subject.

26. The method of claims 3-25, wherein the methylation profiles characteristic of cancer incorporate the position of methylated cytosine with respect to the ends of cfDNA fragments.

27. The method of claims 1-26, wherein genome coverage is about 9x to O.lx;28. The method of claims 3-27, wherein a therapeutic treatment suitable for treatment of the cancer is administered to the subject if the subject is diagnosed with cancer or determined to still have cancer.

29. The method of claims 1-28, wherein the subject is a mammal.

30. The method of claim 29, wherein the mammal is a human.

31. The method of claims 1-30, further comprising generating a report, wherein the report comprises at least one of:(i) the cfDNA fragmentation profile based on the determined cfDNA fragment lengths;(ii) whether the subject has cancer:(iii) whether the level of cfDNA methylation in the subject is altered as compared to the level of cDNA methylation in a healthy subject;(iv) whether the methylation profile is characteristic of a methylation profile in a subject with cancer;(v) whether the cfDNA fragmentation profile is more variable than a reference cfDNA fragmentation profile from a healthy subject; or(vi) whether the methylation profile is characteristic of a methylation profile in a subject with cancer.411623885965.1

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