Methods and compositions for amplifying methylated target DNA molecules

Abstract

The present disclosure provides methods and compositions for preparing DNA molecules useful for determining the methylation status of selected regions of a DNA molecule, which utilize methylation-sensitive restriction enzymes and typically involve the amplification or selective enrichment of one or more target regions.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application Serial No. 63 / 437,016, filed January 4, 2023, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates generally to methods for preparing and analyzing DNA molecules, and more particularly to methods for preparing and analyzing DNA molecules that contain one or more methylation sites. [Background technology]

[0003] Currently, there is a need for improved methods to prepare and quantify low-abundance DNA molecules in samples, such as circulating free DNA (cfDNA), that harbor epigenetic alterations, such as altered methylation status or patterns. Highly sensitive and efficient methods are needed to prepare and quantify low-abundance differentially methylated DNA molecules of interest in samples.

[0004] Improved methods for preparing and quantifying low-abundance DNA molecules are needed, thereby increasing the specificity and sensitivity for distinguishing DNA from different sources, such as tumor DNA and non-tumor DNA. Improved methods for preparing and quantifying low-abundance DNA molecules, such as cfDNA, are needed, thereby enabling the detection of tumor DNA based on epigenetic modifications with improved signal-to-noise resolution. Improved methods for preparing and quantifying low-abundance DNA molecules, such as cfDNA, are needed, thereby improving performance characteristics. Improved methods for preparing and quantifying low-abundance methylated DNA molecules are needed, thereby providing more flexible primer / probe design that is not limited by the location of restriction enzyme recognition sites. Improved methods for preparing and quantifying low-abundance methylated DNA molecules are needed, thereby enabling them to be used for many downstream applications in addition to detecting and / or quantifying methylated DNA molecules of interest.

[0005] While DNA methylation can be detected and analyzed using bisulfite conversion and sequencing, improved methods for preparing and quantifying small amounts of methylated DNA molecules are needed that are not only less destructive to sample DNA molecules but also reduce the cost of the method, for example, by sequencing only the methylated fragments. Improved methods for preparing and quantifying small amounts of methylated DNA molecules are needed that are faster, more cost-effective, and more compatible with sequencing. Summary of the Invention

[0006] To overcome the above-mentioned and other problems in the art, the present disclosure provides a method for determining the methylation status of a selected region of a DNA molecule. The method provided herein utilizes the fact that CpG sites within CpG islands in regulatory regions are predominantly co-methylated in cancer cells, enabling detection of multiple co-methylated CpG sites, in exemplary embodiments, in multiple CpG islands, from low-abundance DNA molecules, such as circulating tumor DNA (ctDNA). The method provided herein can also infer / utilize the methylation status of neighboring CpG sites not within a specific target region, thereby increasing detection specificity and allowing greater flexibility in primer / probe design. The method provided herein allows selective pre-amplification of only fully methylated DNA fragments, thereby enriching DNA molecules of potential interest. The method provided herein improves performance characteristics when distinguishing low-abundance cfDNA of different origins, such as distinguishing ctDNA from cfDNA of non-tumor origin.

[0007] Provided herein, in one aspect, is a method for preparing a deoxyribonucleic acid (DNA) molecule useful for determining the methylation status of a genomic region of interest, the method comprising: a) ligating adaptors to sample DNA molecules obtained or derived from a first sample from a first subject, thereby forming a plurality of adapted DNA molecules, including adapted DNA molecules having a methylation-sensitive restriction enzyme (MSRE) recognition site; b) contacting a plurality of adapted DNA molecules with one or more MSREs, thereby generating MSRE-exposed adapted DNA molecules, wherein adapted DNA molecules exposed to at least a first plurality of MSREs having one or more unmethylated MSRE recognition sites are cleaved by at least one of the MSREs, and adapted DNA molecules exposed to a second plurality of MSREs are not cleaved by any of the MSREs, thereby forming MSRE-exposed uncleaved adapted DNA molecules; c) performing one or more amplification reactions to amplify one or more target regions from the uncleaved adapted DNA molecules or copies thereof exposed to the MSRE, wherein if the one or more target regions are present in the uncleaved adapted DNA molecules exposed to the MSRE, a target region amplicon is formed.

[0008] A target region can be any genomic region of interest or any portion thereof. In some embodiments, the methods disclosed herein provide increased flexibility in selecting or defining a target region. In illustrative examples, a target region can be a genomic region in which epigenetic changes, such as changes in DNA methylation, are associated with or indicate the formation or presence of cancer, including, for example, promoter regions of tumor suppressor genes and, in some embodiments, DNA methylation markers of specific cancer types. In some embodiments, each intended target region amplicon has one or more MSRE recognition sites. In some embodiments, each intended target region amplicon has two or more MSRE recognition sites. In some embodiments, at least 10% of the intended target region amplicons each contain at least two MSRE recognition sites. In some embodiments, at least 15%, 20%, 25%, 30%, or 35% of the intended target region amplicons each contain at least two MSRE recognition sites. In some embodiments, the methods described herein can include at least one predetermined target region amplicon that includes at least 3, 4, 5, 6, 10, 15, 20, 25, 30, 35, or 40 MSRE recognition sites. In some embodiments, the methods described herein can include at least one predetermined target region amplicon that includes between 2 and 45 MSRE recognition sites.

[0009] In some embodiments, the sample DNA molecules are circulating free DNA (cfDNA). In some embodiments, the one or more target region amplicons are generated by one or more polymerase chain reactions (PCR) using one or more primer pairs. In exemplary embodiments, at least one primer in each of the one or more primer pairs is a target-specific primer designed to bind to a specific nucleic acid sequence at or near the target region, typically within the target region. In some embodiments, the one or more target region amplicons are generated using one or more capture probes designed to hybridize to the one or more target regions. In some embodiments, the one or more target region amplicons are generated by one or more ligation target capture (LTC) reactions using one or more probe-dependent primer pairs. In some embodiments, the method further includes detecting the one or more target region amplicons. In some embodiments, the one or more target regions are a set of target regions, and in certain embodiments, the one or more primer pairs are a set of primer pairs, each configured to amplify one target region of the set. In some embodiments, the method further includes quantifying the amount of at least one of the one or more target region amplicons.

[0010] Further details regarding aspects and embodiments of the present disclosure are provided throughout this patent application. The sections and section headings are for ease of reading and are not intended to limit the combinations of the disclosed methods, compositions, and kits, or other functional elements contained therein, across sections. Further details regarding aspects and embodiments of the present disclosure are provided throughout this patent application. The sections and section headings are for ease of reading and are not intended to limit the combinations of the disclosed methods, compositions, and kits, or other functional elements contained therein, across sections. [Brief explanation of the drawings]

[0011] [Figure 1] A representative, non-limiting workflow for preparing DNA molecules with methylation-sensitive restriction enzyme (MSRE) recognition sites is shown. Optional steps are indicated by dashed boxes. In Figure 1, adapter molecules are ligated to DNA fragments obtained or derived from a sample (120). The adapted DNA fragments are then contacted with a mixture of MSREs (130). Targeted amplification (150) or selective enrichment (152) is then performed on the MSRE-treated, adapted DNA molecules to form target region amplicons or enriched subsets of MSRE-treated, adapted DNA molecules containing the target region or a portion of the target region, respectively. An optional step is extraction or isolation of DNA fragments prior to ligation (110). Optionally, a universal amplification step (140) can be, and typically is, performed after MSRE processing and before the targeted PCR or selective enrichment step. Further optionally, the target region amplicons or enriched subsets can be detected and / or optionally quantified (160). [Figure 2] A non-limiting representative workflow for preparing circulating free DNA (cfDNA) from a subject with or suspected of having cancer to determine the methylation status of selected regions of DNA molecules is shown. As shown in Figure 2, the first step involves isolating cfDNA from the subject (111), followed by preparing cfDNA fragments, typically by modifying the cfDNA (115), for adapter ligation. The next step involves ligating adapters to the modified cfDNA fragments (121), followed by contacting the adapter-tagged cfDNA fragments with a mixture of MSREs (130) to form MSRE-processed, adapter-tagged cfDNA fragments. The MSRE-processed, adapter-tagged cfDNA fragments are then amplified by performing a universal amplification step (140). Next, targeted amplification is performed (150), followed by quantifying the target region amplicons using next-generation sequencing (NGS) (161). [Figure 3] A schematic diagram of the reagents and products of the workflow of Figure 2 is shown in Figure 3 (220), which shows the adapted cfDNA fragments of step 121 of Figure 2. As can be seen, the ligated adapters are Y adapters (224). The product of step 130 of the method of Figure 2 is shown in Figure 3 (230), where the MSRE recognition sites containing methylated cytosine bases are not cleaved by the MSRE, while the MSRE recognition sites with unmethylated cytosine bases are cleaved, leaving only the uncleaved sample with the universal primer site intact on the adapter portion of the adapted DNA molecule (232). In 250, targeted PCR primers (258, 262) are used to perform step 150 of Figure 2, amplifying the target region (256, the central region shown in dark shading between and encompassing the binding sites of the targeted PCR primer pair 258, 262) of the universally amplified and MSRE-exposed adapted DNA molecule (252), in which the originally methylated cytosine base ("C" surrounded by an asterisk) is no longer methylated after amplification. The resulting target region amplicon (254) contains additional sequences added from the non-target-specific regions of the targeted PCR primers or additional primers, which are then quantified by sequencing or qPCR (260). Legend: Circled mC: methylated cytosine base; circled C: unmethylated cytosine base; asterisked C: originally methylated cytosine base. [Figure 4] FIG. 1 is a schematic diagram showing different non-limiting locations of PCR primer binding sites on an adaptor-ligated DNA molecule containing a target region (shown with light grey diagonal lines) and methylated CpG sites present both within and outside the target region. [Figure 5A] Figure 1 shows an example workflow, Workflow 1 (W1), for determining the methylation status of sample cfDNA molecules from healthy donors and cancer patients. [Figure 5B]An example workflow, Workflow 2 (W2), for determining the methylation status of sample cfDNA molecules from healthy donors and cancer patients is shown. In Figure 5A (Workflow 1), a nucleic acid sample (411A) is first digested with a mixture of MSREs (430A), followed by library preparation steps including blunt-end repair, adapter ligation, and universal amplification (420A). In the next step, targeted PCR is performed on the library of universally amplified, adapter-ligated, MSRE-treated nucleic acid sample. Figure 5B shows Workflow 2, in which a nucleic acid sample (411B) is first blunt-end repaired and adapter-ligated (420B), followed by digestion with a mixture of MSREs (430B). The adapter-ligated, MSRE-treated sample DNA molecules are then universally amplified in step (440B), followed by targeted PCR (450B). [Figure 6] Schematic diagram of the reagents and products for Workflow 1. DNA fragments are treated with a mixture of MSREs; DNA fragments with methylated MSRE recognition sites are not cleaved, while DNA fragments with unmethylated MSRE recognition sites are cleaved (520A). The MSRE-treated sample is then blunt-end repaired and adapter-ligated (530A). Next, targeted amplification (550A) is performed on the universally amplified, MSRE-treated, adapter-ligated DNA molecule (552A) using a targeted primer pair (558A, 562A). A sample barcode (553A) and NGS sequence (555A) that can be used in downstream NGS reactions can also be added to the amplification product, resulting in an amplified DNA molecule containing the target region flanked by the NGS sequence and sample barcode (554A). The amplified DNA molecule is then quantified and / or sequenced using qPCR (560A). [Figure 7]This is a schematic diagram of the reagents and products of Workflow 2. In contrast to Workflow 1 in Figure 6, Figure 7 shows Workflow 2 as a representative example in which DNA fragments are blunt-end repaired and adapter-ligated (520B) before treatment with MSRE. In this case, adapter-tagged DNA fragments with methylated MSRE recognition sites are not cleaved, while adapter-tagged DNA fragments with unmethylated MSRE recognition sites are cleaved (530B). After universal amplification, only uncleaved adapter-tagged DNA fragments are amplified. The universally amplified and MSRE-treated adapter-tagged DNA molecules (552B) are then targeted using a targeting primer pair (558B, 562B). The amplification products can also be tagged with a sample barcode (553B) and NGS sequence (555B). The resulting amplicons (554B) are then analyzed by sequencing or qPCR (560B). [Figure 8] Comparison of normalized depth of read (DOR) readouts for each MSRE in the MSRE cocktail by Workflow 1 (W1) or Workflow 2 (W2) using unmethylated lambda DNA samples treated with the MSRE cocktail (MSRE) or unmethylated lambda DNA samples treated without the MSRE cocktail (control) is shown. Data for targets containing one or more MSRE recognition sites were extracted and analyzed separately for each MSRE in the cocktail. All DORs were normalized to the median DOR of the methylated pUC19 control. [Figure 9] Figure 1 shows normalized DOR readouts for targeting primer pool 1 of human targets per MSRE recognition site obtained using Workflow 1 (W1) or Workflow 2 (W2) from artificial 100% methylated (100% methyl) or 0% methylated (unmethylated) human gDNA samples (MSRE) treated with the MSRE cocktail or artificial 100% methylated (100% methyl) or 0% methylated (unmethylated) human gDNA samples (control) treated without the MSRE cocktail. All DORs were normalized to the median DOR of the methylated pUC19 control. [Figure 10]Figure 1 shows the normalized DOR readouts for targeting primer pool 2 of human targets per MSRE recognition site obtained using Workflow 1 (W1) or Workflow 2 (W2) from artificial 100% methylated (100% methyl) or 0% methylated (unmethylated) human gDNA samples (MSRE) treated with the MSRE cocktail or artificial 100% methylated (100% methyl) or 0% methylated (unmethylated) human gDNA samples (control) treated without the MSRE cocktail. All DORs were normalized to the median DOR of the methylated pUC19 control. [Figure 11] Figure 1 shows a comparison of DOR heatmaps for targeted primer pool 1 of human targets in cfDNA samples from colorectal cancer (CRC) patients versus healthy donors using Workflow 1. Heatmaps of normalized DORs using targeted primer pool 1 and the Workflow 1 protocol are shown for samples from three individual CRC patients, C1, C2, and C3, and three healthy individuals, H1, H2, and H3. The variant allele frequencies (VAFs) for samples C1, C2, and C3 were 25.31%, 20.52%, and 3.18%, respectively. 100% methylated and unmethylated human gDNA controls are shown in columns 1 and 2, respectively. All DORs were normalized to the median DOR of human non-MSRE controls. [Figure 12] A DOR heatmap comparison of workflow 1 (W1) and workflow 2 (W2) for cfDNA samples from CRC patients versus healthy donors using two targeted primer pools is shown. Samples were analyzed using either W1 or W2, and results from the two pools were combined. The VAF for C1 was 25.31% and the VAF for C2 was 20.52%. All DORs were normalized to the median DOR of the pUC19 control assay. [Figure 13]Figure 1 shows a heatmap comparison of DOR for workflow 1 (W1) and workflow 2 (W2) for cfDNA samples with low VAF (3.19% for W1, 2.51% or 1.78% for W2) from CRC patients versus cfDNA samples from healthy donors using two targeted primer pools. All DORs were normalized to the median DOR of the pUC19 control assay. [Figure 14A] Shown is a DOR heatmap comparison of targeted primer pool 1 (FIG. 14A) on cfDNA samples from 13 CRC patients and 13 healthy donors using workflow 2. All DORs were normalized to the median DOR of human non-MSRE controls. [Figure 14B] A DOR heatmap comparison of targeted primer pool 2 (FIG. 14B) on cfDNA samples from 13 CRC patients and 13 healthy donors using workflow 2. All DORs were normalized to the median DOR of human non-MSRE controls. [Figure 15] Figure 1 shows the correlation between differential methylation levels and the VAF of SNVs. When using the top 40 targets, a higher DOR is associated with a higher VAF of SNVs. [Figure 16A] Normalized DOR in CRC-positive, -negative, and healthy samples is shown. [Figure 16B] Normalized DORs are shown for CRC-positive, -negative, and healthy control samples. The X-axis shows each target in pool 1 (A) and pool 2 (B), and the Y-axis shows the normalized DOR (sample assay DOR / total reads) for each group per target. Target assays on the X-axis were sorted based on the ratio of CRC mean DOR to healthy control mean DOR. The solid line represents the mean normalized DOR, and the shaded area represents ± standard error. [Figure 17A] Normalized DOR among top targets is shown. [Figure 17B]Normalized DOR among top targets is shown. Samples are grouped by SNV VAF range. (A) The top 10 targets (ranked by Gini importance) were used to group CRC samples with various SNV VAF ranges vs. healthy control samples. (B) The top 20 targets (ranked by Gini importance) were used to group CRC samples with various SNV VAF ranges vs. healthy control samples. The solid line represents the mean normalized DOR, and the shaded area is ± standard error. [Figure 18A] Heatmap showing the comparison of normalized DOR between healthy / negative and CRC samples for the top targets in pool 1. [Figure 18B] Heatmap showing the normalized DOR comparison between healthy / negative samples and CRC samples for the top targets in pool 2. (A) Each column is for one sample, and each row represents a target in the top 10 assays. (B) Each column is for one sample, and each row represents a target in the top 20 assays. Values ​​are normalized DOR. [Figure 19A] 1 shows the ROC curves of the test set for different target groups. [Figure 19B] 1 shows the ROC curves of the test set for different target groups. [Figure 19C] 1 shows the ROC curves of the test set for different target groups. [Figure 19D] ROC curves for the test set for different target groups are shown. Random forest models were generated using (A) all targets, (B) the top 20 targets from pool 1 and pool 2, (C) the top 20 targets from pool 1 only, and (D) the top 20 targets from pool 2 only. Target ranking was based on the ratio of mean DOR for CRCs to mean DOR for healthy controls. [Figure 20] The number of MSRE cleavage sites per assay associated with the DOR ratio in CRC / healthy controls is shown. The CRC / healthy control ratio was calculated as mean DOR (CRC) / mean DOR (healthy controls), and the ratios are ranked from highest to lowest DOR ratio. Highest DOR ratio = Rank 1. [Figure 21]The MSRE+ Hybrid Capture workflow is outlined. The nucleic acid sample (411C) is first blunt-end repaired and adapter-ligated (420C), followed by digestion with a mixture of MSREs (430C). The adapter-ligated, MSRE-treated sample DNA molecules are universally amplified in step (440C), followed by barcoding PCR (460C). The libraries are normalized and pooled (470C), then captured and amplified using the MSRE HC panel. [Figure 22] Normalized DORs are shown for CRC-positive, -negative, and healthy control samples. The X-axis shows each target, and the Y-axis shows the normalized DOR for each group per target. Target assays on the X-axis were sorted based on the ratio of the mean CRC DOR to the mean healthy control DOR. The solid line represents the mean normalized DOR, and the shaded area represents ± standard error. All DORs were normalized to the median DOR of human non-MSRE controls. [Figure 23A] Normalized DOR for top targets is shown. [Figure 23B] Normalized DOR for top targets is shown. Samples are grouped by SNV VAF range. (A) The top 20 targets (ranked by CRC / healthy DOR ratio) were used to group CRC samples vs. healthy samples with various SNV VAF ranges. (B) The top 40 targets (ranked by CRC / healthy DOR ratio) were used to group CRC samples vs. healthy samples with various SNV VAF ranges. The solid line is the mean normalized DOR, and the shaded area is ± standard error. [Figure 24A] A heatmap is shown for comparing normalized DOR between healthy / negative and CRC samples. [Figure 24B] Heatmaps are shown comparing normalized DOR between healthy / negative samples and CRC samples. Each column represents a sample, and each row represents a target in the top 20 (Figure 24A) or top 40 (Figure 24B) assays. Values ​​are normalized DOR. Across targets, CRC samples can be distinguished from negative and healthy samples. [Figure 25A] ROC curve for the test set is shown. The workflow for MSRE+Hybrid Capture. A random forest model was generated using the top 40 targets with high DOR ratios. [Figure 25B] ROC curves for the test set are shown. MSRE+mPCR workflow. A random forest model was generated using the top 20 targets with high DOR ratios. [Figure 26] The number of MSRE cleavage sites per assay associated with the CRC / healthy DOR ratio is shown. Not all assays with multiple MSRE cleavage sites provided a high, discriminatory CRC / healthy DOR ratio. [Figure 27A] Shows good targets common to HC and mPCR. [Figure 27B] Good targets common to HC and mPCR are shown. A) Cross-checking the top 40 targets using HC with the top 40 targets from previous mPCR studies (Examples 1 and 2) using the same target set reveals that 24 / 40 HC targets were found in both mPCR studies, and 36 / 40 were found in at least one mPCR study. B) Of the top 24 common good target assays, 23 / 24 targets contain 10 or more cleavage sites.

[0012] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. "Comprising / comprising A or B" means including A, or B, or A and B. Furthermore, it should be understood that all base sizes or amino acid sizes and all molecular weight or molecular mass values ​​given for DNA molecules or polypeptides are approximate and are provided for illustrative purposes.

[0013] Ranges provided herein are understood to be shorthand for all of the values ​​within that range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 1-49, 1-25, 1.7-31.9, etc. (including fractions thereof unless the context clearly dictates otherwise). Any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the recited range, and, where appropriate, fractions thereof (such as integer tenths and hundredths), unless otherwise indicated. Also, any numerical range recited herein for any physical characteristic, such as polymer subunits, size, or thickness, should be understood to include any integer within the recited range, unless otherwise indicated. When a range is given that includes multiple lower values ​​and multiple upper values, one of ordinary skill in the art will recognize that the selected range includes lower values ​​that are less than the upper value.

[0014] As used herein, "about" or "consisting essentially of" means ±10% of the stated range, value, or structure, unless otherwise indicated. As used herein, the terms "include" and "comprise" are used interchangeably. As used herein, "comprising" is synonymous with "including," "containing," or "characterized by" and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. In each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms. The invention(s) illustratively described herein may suitably be practiced in the absence of any element(s), limitation(ies) not specifically disclosed herein.

[0015] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below.All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.In the event of any discrepancy with other descriptions, the descriptions in this specification, including explanations of terms, shall prevail.In addition, the materials, methods, and examples are merely illustrative and are not intended to be limiting.

[0016] It is understood that certain features of aspects and embodiments herein that are discussed for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various aspects and embodiments that are discussed for brevity in the context of a single aspect or embodiment may also be provided separately or in any suitable subcombination. All combinations of aspects and embodiments are specifically embraced and disclosed herein as if each and every combination were individually expressly disclosed. In addition, all subcombinations of various aspects and embodiments and elements thereof are also specifically disclosed herein, even if each and every such subcombination is not individually expressly disclosed herein. DETAILED DESCRIPTION OF THE INVENTION

[0017] Both global and local epigenetic alterations are widely recognized as hallmarks of cancer. These alterations include global decreases in CpG methylation levels and discrete regions of hypermethylation in CpG islands, typically located in the promoter regions of tumor-suppressor genes. Hypermethylation has been associated with cancer progression and the silencing of growth-regulatory and tumor-suppressor genes. Consequently, DNA methylation biomarkers are being utilized to develop novel assays for monitoring cancer progression, treatment response, and early detection. The presence of cancer can be detected by analyzing the methylation status of specific CpG sites that are predominantly methylated in DNA from tumor cells, including circulating tumor DNA (ctDNA) released from tumor cells.

[0018] The present disclosure addresses many long-standing needs and long-standing problems in the art, including but not limited to those mentioned in the "Background Art" section of this specification.For example, provided herein is a method for preparing deoxyribonucleic acid (DNA) molecules useful for determining the methylation status of genomic regions of interest.Such method can be used to detect methylated DNA molecules of interest, and demonstrates better specificity for detecting methylated DNA molecules of interest and improved signal-to-noise resolution for ctDNA samples with low VAF.

[0019] Thus, as shown in Figure 1, provided herein, in some embodiments, are methods for preparing deoxyribonucleic acid (DNA) molecules having a methylation-sensitive restriction enzyme (MSRE) recognition site. Such methods are useful, for example, for determining the methylation status of a genomic region of interest, for detecting methylated DNA molecules, for quantifying methylated DNA molecules, and / or for detecting ctDNA. Steps of such embodiments are shown in boxes in Figure 1, with optional steps in such embodiments being shown in dashed boxes. Such methods, in non-limiting examples, can include the following steps: a) ligating adapters to sample DNA molecules obtained or derived from a first sample from a first subject (120), thereby forming adapted DNA molecules comprising adapted DNA molecules having a methylation-sensitive restriction enzyme (MSRE) recognition site; b) contacting the plurality of adapted DNA molecules with one or more MSREs (130), thereby generating MSRE-exposed adapted DNA molecules, wherein the adapted DNA molecules exposed to at least a first plurality of MSREs having one or more unmethylated MSRE recognition sites are cleaved by at least one of the MSREs, and the adapted DNA molecules exposed to a second plurality of MSREs are not cleaved by any of the MSREs, thus forming MSRE-exposed uncleaved adapted DNA molecules; c) performing one or more amplification reactions (140, 150) to amplify one or more target regions from the uncleaved adapted DNA molecule or a copy thereof exposed to the MSRE, wherein if the one or more target regions are present in the uncleaved adapted DNA molecule exposed to the MSRE, a target region amplicon is formed.

[0020] Typically, target region amplicons are generated by one or more polymerase chain reactions (PCRs) (140, 150) on uncleaved, adapted DNA molecules exposed to an MSRE. More specifically, typically, one or more PCRs are performed using one or more primer pairs, where at least one primer in each of the one or more primer pairs is a target-specific primer designed to bind to a specific nucleic acid sequence at or near, typically within, the genomic region of interest (150). Typically, one or more target region amplicons are detected. More specifically, one or more of the one or more target regions are a set of target regions, and the one or more primer pairs are a set of primer pairs, each configured to amplify one target region of the set. Alternatively, or in addition, typically after the universal amplification step (140), one or more subsets of uncleaved, adapted cfDNA exposed to an MSRE having one or more target regions can be enriched using one or more hybrid capture probes that bind to a nucleic acid sequence at or near, typically within, the target region (152). Typically, one or more target region amplicons can be enriched. More specifically, one or more of the one or more target regions is a set of target regions, and one or more hybrid capture probes is a set of hybrid capture probes, each configured to enrich one target region of the set.

[0021] Another exemplary method herein for preparing a DNA molecule useful for determining the methylation status of a selected region of a DNA molecule comprises the following steps: a) ligating adapters to circulating free DNA (cfDNA) obtained or derived from a first liquid sample, thereby forming a plurality of adapted cfDNAs, including adapted cfDNAs having MSRE recognition sites; b) contacting the plurality of adapted cfDNAs with one or more MSREs, thereby generating MSRE-exposed adapted cfDNAs, wherein the MSRE-exposed adapted cfDNAs are not cleaved by the one or more MSREs, thereby forming MSRE-exposed uncleaved adapted cfDNAs; c) performing an amplification step to amplify a set of target regions from the amplified and MSRE-exposed uncleaved adapted cfDNA, each target region having one or more MSRE recognition sites, wherein a target region of the set is amplified if the target region is present in one or more of the amplified and MSRE-exposed uncleaved adapted cfDNA, thereby generating a target region amplicon; and d) quantifying the amount of at least some of the target region amplicons by performing a next-generation sequencing (NGS) reaction on the clonally amplified target region amplicons or amplicons derived therefrom. In exemplary embodiments, such quantification provides a quantitative value for methylated target regions. Some, most, a very large majority, almost all, or all of the unmethylated target regions are not amplified in the targeted amplification reaction because they are digested with MSRE prior to targeted amplification.

[0022] The methods herein can optionally include extracting or isolating DNA molecules from a subject (110), for example, from a liquid sample within the subject, which in exemplary embodiments includes cfDNA. Alternatively, DNA molecules extracted from or derived from the subject can be fragmented, for example, by shearing, before being treated with the exemplary methods described herein. In certain exemplary embodiments, cfDNA is extracted from or derived from a subject who has, is suspected of having, or has had cancer. In some embodiments, methylation of genomic DNA is optionally analyzed in the tumor of such a subject using the methods herein, and methylation of cfDNA is analyzed, typically from the same subject, using the methods herein, to identify ctDNA in the cfDNA sample.

[0023] Thus, a non-limiting exemplary embodiment of a method for preparing a DNA molecule herein is shown in Figure 2, and the corresponding DNA reactants and products for such a method are shown in Figure 3. Such a non-limiting exemplary embodiment comprises the following steps: a) extracting circulating free DNA (cfDNA) from a liquid (e.g., plasma) sample from a subject (111); b) modifying cfDNA from the sample for adapter ligation by preparing a cfDNA derivative (115); and forming a plurality of adapted cfDNAs, including adapted cfDNAs (220) having one, two, three, four, or more MSRE recognition sites (one MSRE recognition site (225) is shown), by ligating adapters to the resulting cfDNA derivatives (121). c) contacting the plurality of adapted cfDNAs with one or more MSREs (130), thereby generating MSRE-exposed adapted cfDNAs, wherein the plurality of MSRE-exposed adapted cfDNAs are not cleaved by the one or more MSREs, thereby forming MSRE-exposed uncleaved adapted cfDNAs (232); d) performing a first amplification step (140) using universal primers, wherein uncleaved, adapted cfDNA exposed to at least a plurality of nMSREs is amplified (252); e) performing a second amplification step to amplify a set of target regions (150) from the amplified and MSRE-exposed uncleaved adapted cfDNA (252), wherein each target region (the central region (256) shown in dark shading between and encompassing the binding sites of the targeted PCR primer pair (258, 262)) has one or more MSRE recognition sites, and if the target region is present in one or more of the amplified and MSRE-exposed uncleaved adapted cfDNA, the target region of the set is amplified, thereby generating target region amplicons (254); f) detecting and / or quantifying the amount of at least some of the target region amplicons of the set by performing a next generation sequencing (NGS) reaction (161, 260) or a qPCR reaction (260) on the clonally amplified target region amplicons or amplicons derived therefrom.

[0024] In exemplary embodiments, such as those shown in FIG. 2, the methods described herein can include modifying (115) extracted or isolated DNA molecules (e.g., cfDNA) before adapters are ligated to the modified DNA molecules (e.g., modified cfDNA) to generate DNA molecules (e.g., modified DNA molecules, such as modified cfDNA) derived from the sample DNA molecules (e.g., cfDNA). Such steps can include preparing the sample DNA molecules for adapter ligation (e.g., steps that can be performed during NGS library preparation). For example, such steps can include blunt-end repair and / or A-tailing, including the addition of a polyA tail or a single A tail (FIG. 2 (115)). In exemplary embodiments, the adapters used for ligation are Y adapters (222), such as those used in NGS library preparation. Typically, the adapters do not contain MSRE recognition sites.

[0025] In certain embodiments, the adapted cfDNA (220) has two, three, four, or more MSRE recognition sites. For example, in some embodiments, the adapted cfDNA may have 3, 4, 5, 6, 10, 15, 20, 25, 30, 35, 40, or more MSRE recognition sites. The adapted cfDNAs (222, 223) in Figure 3 each contain an MSRE recognition site (225). The MSRE recognition site in the upper adapted cfDNA (222), which in this example is ctDNA, contains a CpG site with a methylated cytosine residue (shown as a circled "mC"), which prevents MSRE cleavage. In contrast, the CpG site in the MSRE recognition site in the lower adapted cfDNA (223) is unmethylated (shown as a circled C).

[0026] Thus, when adaptor-attached cfDNA with one or more MSREs that recognize MSRE recognition sites containing CpG sites (mC or C in Figure 3) is contacted, adaptor-attached cfDNA / ctDNA with only methylated MSRE recognition sites remains uncleaved (232), whereas some, many, most, almost all, or all of the adaptor-attached cfDNA with one or more unmethylated MSRE recognition sites is cleaved (233), and therefore, such cleaved DNA molecules are not amplified during the subsequent amplification reaction.

[0027] The methods described herein typically involve performing PCR using one or more targeted primer pairs, each including at least one primer designed to bind to a specific sequence at or near a target region of the sample nucleic acid, typically within the target region, to amplify one or more target regions of an adapted DNA molecule exposed to an MSRE. The targeted PCR primer pair typically defines the ends of the target region amplicon. As discussed in more detail herein, Figure 4 provides various embodiments illustrating the location of primer binding sites on a DNA molecule subjected to exemplary methods described herein. In some embodiments, the one or more target regions are a set of target regions, and the one or more primer pairs are a set of primer pairs, each configured to amplify one target region of the set.

[0028] In the exemplary methods described herein, at least one primer (e.g., 258) of each pair of primers (e.g., 258 and 262) used in targeted amplification (e.g., PCR) or in additional PCR(s) performed on the amplicons generated by the targeted PCR can optionally include a sample barcode / index. Furthermore, such primers for the targeted PCR or additional PCR(s) can be designed to contain sequences that can be utilized in a subsequent sequencing step (i.e., "NGS sequencing"). For example, such sequences can include NGS flow cell binding sites (e.g., Illumina P5 and P7 sequencing) and / or NGS sequencing primer binding sites. Thus, primers used in the targeted PCR or additional PCR(s) may be designed to include NGS sequences and / or sample barcodes / indexes that can be used for downstream NGS analysis.

[0029] In certain exemplary embodiments, primers may be designed as probe-dependent primers, in which a targeting probe designed to bind to a specific nucleic acid sequence at or near a region of interest is physically linked to a universal primer designed to bind to a universal primer binding sequence on the adapter portion of an adapted DNA molecule (see, e.g., Pel, et al., "Rapid and highly-specific generation of targeted DNA sequencing libraries enabled by linking capture probes with universal primers," PLoS ONE 13(12):e0208283 (2018), incorporated herein by reference in its entirety). Primers in probe-dependent primers may also be designed to include an NGS sequence and / or sample index that can be used for downstream NGS analysis.

[0030] In some embodiments, the method further includes detecting and / or quantifying target region amplicons, e.g., by sequencing or qPCR, thereby determining the methylation status of one or more MSRE recognition sites within or near one or more target regions on the sample DNA molecule (e.g., cfDNA). For example, an NGS reaction can be performed on clonally amplified target region amplicons generated by performing an additional amplification reaction to amplify at least some of the target region amplicons; in some embodiments, this additional amplification reaction is a clonal amplification reaction, e.g., on an NGS substrate, to form clonally amplified target region amplicons. Furthermore, primers used to generate amplicons in any step, including, for example, primers (e.g., 262 and 258) used in the targeted amplification step, can contain additional sequences, such as NGS sequences, or common sequences that are binding sites for one or more additional primer sets used in one or more additional PCR rounds to amplify the products of the targeted amplification and generate additional amplicons. Such additional sets of primers capable of binding to sequences appended to the targeted amplicon product can include additional sequences, such as NGS sequences, to generate amplicons containing additional sequences surrounding the targeted region, as shown in amplification product (254) in Figure 3. Such targeted amplification(s) and / or additional amplification(s) can be performed in successive cycling reactions or within the same cycling reaction, and in an exemplary embodiment, are performed before the NGS clonal amplification reaction.

[0031] As provided in the illustrative example of Figure 2, the methods described herein can optionally include one or more universal / library pre-amplification and / or amplification steps before and / or after the targeted amplification step (Figure 2 (140)). Such methods include performing one or more PCR reactions using universal primers designed to bind to primer binding sites on the adapter portions of the adapted DNA molecules and / or universal primer binding sites on the primers used for the targeted amplification(s) or optional additional amplification(s).

[0032] In exemplary embodiments of the methods described herein, including but not limited to the method of Figure 2, the liquid sample is a blood, plasma, serum, or urine sample, which typically contains cfDNA. In exemplary embodiments, the methylation status of the set of target regions indicates the presence or absence of ctDNA in the cfDNA sample, which typically indicates the presence or absence of cancer in the subject.

[0033] Sample extraction and enrichment of DNA molecules The sample useful in the methods herein can be virtually any nucleic acid sample. In exemplary embodiments, the nucleic acid sample can be extracted or isolated from a subject. Methods for extracting or isolating DNA molecules from samples such as tissue samples, and in exemplary embodiments described herein, liquid samples, are known. Particularly useful methods in representative embodiments include methods for isolating circulating free DNA (cfDNA) from liquid samples, in exemplary embodiments, from blood, serum, urine, vitreous, sputum, saliva, tears, sweat, feces, bile, lymph, cervical mucus, or semen samples, and in exemplary embodiments, from plasma samples.

[0034] In certain exemplary embodiments, extracted or isolated DNA molecules can be concentrated. Reagents, kits, and related methods for isolating and concentrating nucleic acids from biological samples, including isolating cfDNA from liquid samples, are generally commercially available. For example, isolating cfDNA from a liquid (e.g., a blood or blood-derived sample, such as a serum or plasma sample) can involve binding DNA molecules from the sample to a matrix and isolating the DNA molecules in the presence of a solvent. In some embodiments, the method further includes incubating the biological sample containing the DNA molecules with a protease before contacting the DNA molecules with the matrix. In some embodiments, the method can further include washing the matrix with a wash buffer to remove impurities, and optionally drying the matrix. The concentrated nucleic acid sample can be eluted from the matrix using an elution buffer.

[0035] Other methods for nucleic acid isolation, such as cfDNA isolation and optional enrichment of certain cfDNA, can include microfluidic devices, such as ion exchange columns or solid-phase isolation based on DNA capture by immobilized beads or functionalized surfaces. Other methods include liquid-phase isolation using electric fields or chemical reagents instead of functionalized surfaces. Typically, in the embodiments described herein, cfDNA isolation from patient samples is performed using a DNA isolation kit (e.g., QIAamp Circulating Nucleic Acid Kit (Qiagen)). In some embodiments, hybridization capture using hybrid capture probes is used to preferentially enrich DNA. For example, a probe is used that binds to a specific nucleic acid sequence at or near the target region, usually within the target region.

[0036] In some embodiments, cfDNA or its amplicons of a certain size can be enriched before or after subjecting the cfDNA to the methods described herein. In some embodiments, size selection can be performed before sequencing library preparation. In some embodiments, size selection can be performed after sequencing library preparation and before sequencing. In some embodiments, size selection is performed on the sequencing-ready pool. The enriched cfDNA molecules can be, for example, 50-1200 base pairs in length, 70-500 base pairs in length, 100-200 base pairs in length, or 130-170 base pairs in length. In some embodiments, the enriched cfDNA molecules are 50-200 bp in length. In some embodiments, the enriched cfDNA molecules are 60-200 bp in length, 60-150 bp in length, or 60-100 bp in length before the enriched cfDNA molecules or derivatives thereof are ligated to adapters in the methods described herein. In some embodiments, the enriched cfDNA molecules have a length of less than 150, 100, 90, 75, or 50 before they are ligated to adapters. Such enrichment methods can be performed, for example, using the method of WO2018156418 A1, Stray, et al. (incorporated herein by reference in its entirety).

[0037] In some embodiments, the sample is enriched with tumor DNA molecules, and these tumor DNA molecules are usually less than 160bp, with a peak length of about 145bp.In exemplary embodiments, the enriched nucleic acid sample is circulating tumor DNA (ctDNA) or its amplicon.In such embodiments, the enriched nucleic acid is less than 160, 150, 145, 120, 100, 90, 75, or 50bp in length.In some embodiments, size selection is used to filter out cfDNA molecules (usually longer than ctDNA molecules, about 165bp) that are not derived from tumors but are derived from clonal hematopoiesis (CHIP) of undetermined significance.

[0038] DNA methylation biomarkers are increasingly being utilized in the development of novel assays for use in women's health and / or organ health. For example, methylation profiling is being used in noninvasive prenatal testing (NIPT) to monitor epigenetic changes in the placenta and fetus, and for the asymptomatic detection of preterm birth, preeclampsia, placental insufficiency, and fetal growth restriction. Methylation markers can also indicate congenital fetal disorders. Furthermore, methylation biomarkers are also used for organ health monitoring, such as predicting and monitoring organ rejection in transplant patients and monitoring immune changes in transplant rejection, as well as for monitoring organ health in high-risk or predisposed individuals. Thus, the samples described herein may be maternal samples, fetal samples, or samples from transplant patients.

[0039] In some embodiments, the sample is enriched for fetal DNA molecules. In exemplary embodiments of such embodiments, the enriched nucleic acid sample is fetal circulating free DNA or its amplicon. In such embodiments, the length of the enriched nucleic acid is 100 bp to 220 bp. In some embodiments, the length of the fetal cfDNA nucleic acid sample is in the range of 100 bp to 200 bp, 120 bp to 180 bp, 140 bp to 160 bp, 150 bp to 170 bp, 160 bp to 190 bp, or 170 bp to 220 bp.

[0040] In some embodiments, the sample is enriched for DNA molecules derived from the transplant donor. In exemplary embodiments of such embodiments, the enriched nucleic acid sample is circulating free DNA or amplicons thereof derived from the transplant donor. In such embodiments, the length of the enriched nucleic acid is 100 bp to 220 bp. In some embodiments, the length of the nucleic acid sample of transplant donor cfDNA is in the range of 100 bp to 200 bp, 120 bp to 180 bp, 140 bp to 160 bp, 150 bp to 170 bp, 160 bp to 190 bp, or 170 bp to 220 bp.

[0041] Subject The subject used in the methods described herein can be virtually any animal, but in exemplary embodiments is a mammal, and in even more exemplary embodiments is a human. In some embodiments, the subject is suspected of or at risk of having a disease (in some embodiments, cancer). In some embodiments, the subject is a pregnant woman. In some embodiments, the subject is a subject that includes an organ from another individual.

[0042] In embodiments in which the subject is afflicted with cancer, the cancer can be any type of cancer, provided that the genome of the subject's cancerous cells has portions of their genome that are differentially methylated compared to the subject's non-cancerous cells. Typically, some, most, nearly all, or all of the subject's cancer cells have regions of their genome that are unmethylated or more methylated than the subject's non-cancerous cells. Thus, in some embodiments, the subject is afflicted with ovarian cancer, soft tissue sarcoma, peripheral T-cell carcinoma, colorectal cancer, intrahepatic cholangiocarcinoma, glioblastoma, esophageal cancer, cutaneous T-cell lymphoma, non-Hodgkin's lymphoma, urothelial carcinoma, basal cell carcinoma, epithelioid sarcoma, pancreatic cancer, non-small cell lung cancer, Hodgkin's lymphoma, renal cell carcinoma, mesothelioma, metastatic uveal melanoma, kidney cancer, blood cancer, HER2-expressing cancer, non-melanoma skin cancer, liposarcoma, hepatocellular carcinoma, or hepatocellular carcinoma. , small lymphocytic lymphoma, prostate cancer, breast cancer, anal cancer, marginal zone lymphoma, cutaneous squamous cell carcinoma, thyroid cancer, medullary thyroid cancer, triple-negative breast cancer, neuroendocrine prostate cancer, bladder cancer, paraganglioma, medulloblastoma, superficial basal cell carcinoma, head and neck squamous cell carcinoma, hematological malignancies, melanoma, B-cell lymphoma, relapsed / refractory acute myeloid leukemia, angiosarcoma, osteosarcoma, refractory cervical cancer, cholangiocarcinoma, osteosarcoma, biliary tract cancer, castration-resistant Prostate cancer, gastroesophageal adenocarcinoma, rhabdomyosarcoma, carcinoma, non-muscle invasive bladder cancer, uveal melanoma, small cell lung cancer, cervical cancer, primary open-angle glaucoma, follicular lymphoma, synovial sarcoma, liver cancer, carcinosarcoma, meningeal brain tumor, T-cell lymphoma, lymphoma, small cell lung cancer, mantle cell lymphoma, B-cell malignancies, endometrial cancer, myxoid / round cell liposarcoma, metastatic Merkel cell carcinoma, neuroblastoma, chronic lymphocytic leukemia, Affected by one or more cancers (e.g., one cancer), including giant cell tumor of tendon sheath, sarcoma, acute myeloid leukemia, skin cancer, nasopharyngeal carcinoma, relapsed / refractory Ewing's sarcoma, bone cancer, glioma, salivary gland cancer, gastric cancer, benign tumors, low-grade serous ovarian cancer, metastatic breast cancer, multiple myeloma, diffuse large B-cell lymphoma, relapsed / refractory lymphoma, metastatic colorectal cancer, advanced malignancies, and acute lymphoblastic leukemia.

[0043] In certain embodiments of the methods described herein, the subject is suffering from cancer of the abdomen or abdominal wall, adrenal gland, anus, appendix, bladder, bone, brain, breast, cervix, chest wall, colon, diaphragm, duodenum, ear, endometrium, esophagus, fallopian tube, gallbladder, gastroesophageal junction, head and neck, kidney, larynx, liver, lung, lymph node, malignant effusion, mediastinum, nasal cavity, omentum, ovary, pancreas, pancreaticobiliary region, parotid gland, pelvis, penis, pericardium, peritoneum, pleura, prostate, rectum, salivary gland, skin, small intestine, soft tissue, spleen, stomach, thyroid, tongue, trachea, ureter, uterus, vagina, vulva, or Whipple resection site. In exemplary embodiments, the cancer is selected from nasopharyngeal carcinoma, hepatocellular carcinoma, breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, lung cancer, esophageal cancer, prostate cancer, bladder cancer, melanoma, and acute leukemia. In an exemplary embodiment, the cancer is selected from colorectal cancer.

[0044] Detection and Analysis The methods described herein typically involve detecting and, optionally, quantifying nucleic acids, including DNA, cfDNA, amplified and MSRE-exposed cfDNA with uncleaved adapters, enriched subsets of DNA containing target regions, and, in exemplary embodiments, target region amplicons or amplicons derived therefrom. In some embodiments, cfDNA from a blood sample from an individual is analyzed. Without being limited by theory, it is believed that cfDNA is released when certain cells, such as cancer cells, undergo, for example, necrosis or apoptosis. In some embodiments, the methods described herein can be used to detect methylation in target regions or nucleic acid sequences of interest that are present in a small percentage of DNA in samples, such as cfDNA from fetuses, cells from donated organs, or, in exemplary embodiments, from cancer cells.

[0045] In the methods described herein, at least some of the subsets of uncleaved adaptor-attached cfDNA amplified and exposed to an MSRE can be enriched using a set of hybrid capture probes to form at least some of the enriched subsets before detecting or quantifying the amount of at least some of the enriched subsets of uncleaved adaptor-attached cfDNA amplified and exposed to an MSRE. In methods that include detecting or quantifying target region amplicons, such target region amplicons can be enriched using a set of hybrid capture probes before being detected or quantified. In exemplary embodiments, the methods described herein can include detecting or quantifying target region amplicons without a selective enrichment step.

[0046] In some embodiments, the method further comprises an additional amplification reaction that amplifies at least some of the amplified target region amplicons or amplifies at least some of the enriched subset of uncleaved adaptor-attached cfDNA that has been amplified and exposed to the MSRE for detection and quantification. The additional amplification reaction can be, for example, a quantitative PCR (qPCR reaction) such as a TAQMAN assay (LIFE TECHNOLOGIES) or an INVADER assay (THIRD WAVE TECHNOLOGIES), digital PCR, or any other method for detecting and / or quantifying target DNA, which typically includes a target region that includes one or more MSRE sites, as used herein.

[0047] In some embodiments, the additional amplification reaction is a clonal amplification reaction to form clonally amplified target region amplicons or to form a clonally amplified enriched subset of the amplified and MSRE-exposed uncleaved adaptored cfDNA. To detect or quantify at least some of the amplified target region amplicons and / or selectively enriched target region amplicons, the methods described herein can include: i) performing an additional amplification reaction to amplify at least some of the amplified target region amplicons, where the additional amplification reaction is a clonal amplification reaction to form clonally amplified target region amplicons; and ii) performing a next-generation sequencing reaction on the clonally amplified target region amplicons. To detect or quantify at least some of the enriched subset of amplified and MSRE-exposed uncleaved adapted cfDNA, the methods described herein can include: (i) performing an additional amplification reaction to amplify at least some of the enriched subset of amplified and MSRE-exposed uncleaved adapted cfDNA, where the additional amplification reaction is a clonal amplification reaction to form a clonally amplified and enriched subset of amplified and MSRE-exposed uncleaved adapted cfDNA; and (ii) performing a next-generation sequencing reaction on the clonally amplified and enriched subset of amplified and MSRE-exposed uncleaved adapted cfDNA. The detecting or quantifying method described herein can include performing a sequencing reaction on the clonally amplified target region amplicons.

[0048] In some embodiments, the sequencing reaction is a next-generation sequencing reaction. In the methods described herein, detecting or quantifying includes counting sequence reads generated from clonally amplified target region amplicons. Quantifying can also include determining the read depth for each target region for at least a portion of the target region. The read depth for each target region can be normalized to the read depth of a normalization sequence. The DNA sequence used for normalization can be derived from genomic DNA or a control plasmid and can contain no MSRE cleavage sites or fully methylated MSRE cleavage sites, depending on experimental conditions such as the selection of MSREs. The DNA sequence used for normalization for quantitative methods described herein, such as NGS, can be 100% methylated at the MSRE cleavage sites or have no MSRE cleavage sites. The normalization sequence can be a 100% methylated artificial DNA molecule derived from a control genomic DNA sample or a pUC19 control plasmid. The normalization sequence can be derived from a lambda control plasmid with 100% methylated DNA. For example, a fully methylated (100% methylated) synthetic sequence can also be considered a normalization sequence. In some embodiments, a normalization sequence can be a non-MSRE DNA molecule that does not have an MSRE recognition site for any of the MSRE(s) used. In some embodiments, a non-MSRE sequence for normalization does not have an MSRE recognition site on the intended amplicon. In some embodiments, a non-MSRE sequence for normalization does not have an MSRE recognition site on the intended amplicon and within + / - 200 bp, + / - 150 bp, + / - 100 bp, + / - 50 bp, or + / - 40 bp. Such a non-MSRE DNA molecule can be derived from a first subject. A non-MSRE DNA molecule according to the methods described herein can be a synthetic sequence. A normalization sequence can be a spike-in control DNA sample that does not undergo an MSRE digestion step.The spike-in control DNA sample can be a fully methylated genomic, plasmid, or synthetic DNA sample. In some embodiments, the spike-in control DNA sample used for normalization can be 0.00005%, 0.0001%, 0.005%, 0.001%, 0.05%, 0.1%, 0.2%, 0.5%, 0.7%, 0.8%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, or more fully methylated DNA by mass. The spike-in control sample can be, by weight, 0.00005-2%, 0.0001-2%, 0.005-2%, 0.001-2%, 0.05-2%, 0.1-2%, 0.5-2%, 1-2%, 0.00005-1.5%, 0.00005-1.2%, 0.00005-1%, 0.00005-0.8%, 0.00005-0.5%, 0.00005-0.3%, or 0.00005-0.2% fully methylated or non-MSRE DNA. The methods described herein can include two or more control or spike-in control samples, e.g., 2, 3, 4, 5, or more. DNA sequencing technologies, particularly high-throughput next-generation sequencing technologies (often referred to as massively parallel sequencing technologies), such as those employed in MYSEQ (ILLUMINA), HISEQ (ILLUMINA), ION TORRENT (LIFE TECHNOLOGIES), GENOME ANALYZER ILX (ILLUMINA), and GS FLEX+ (ROCHE 454), can be used to quantitatively measure the number of copies of a target region present after MSRE digestion, for example, but not limited to, clonally amplified target region amplicons or clonally amplified and enriched subsets of uncleaved adapter-attached cfDNA amplified and exposed to MSRE, thus providing quantitative information regarding the number and / or amount of methylation in sample DNA molecules, e.g., cfDNA. High-throughput gene sequencers can be adapted to use barcoding (i.e., sample tagging with a distinctive nucleic acid sequence) to identify specific samples from an individual, thereby enabling simultaneous analysis of multiple samples in a single DNA sequencer run.The number of times (reads) a given region of the genome is sequenced in a library preparation (or other nucleic acid preparation of interest) will be proportional to the number of copies of that sequence that were not digested (i.e., methylated) by MSRE. Methods such as those described herein that utilize NGS detection can, in some embodiments, have an average read depth of at least 200, 500, 1000, 2000, 2900, 3000, 3500, 4000, 5000, 10,000, 50,000, 75,000, 100,000, 130,000, 150,000, 175,000, or 200,000.

[0049] The methods described herein can include analyzing data obtained from next-generation sequencing technology. In some embodiments of the methods described herein, clonally amplified target region amplicons, or a clonally amplified and enriched subset of uncleaved adapter-attached cfDNA amplified and exposed to MSRE, can be sequenced using next-generation sequencing technology. Nucleic acid sequencing data can be generated for amplicons created by PCR, for example, multiplex targeted PCR (mPCR). In some embodiments, the multiplex PCR can be tiled multiplex PCR. Those skilled in the art have access to algorithm design tools that can be used and / or adapted to analyze sequencing data. In addition, those skilled in the art can determine appropriate parameters for measuring alignment to consensus sequences and / or known target region sequences, including any algorithms necessary to achieve maximum alignment across the entire length of the sequences being compared.

[0050] Sequencing reads can be demultiplexed using in-house tools and mapped in single-end mode using the mem function of the Burrows-Wheeler alignment software, BWA (Burrows-Wheeler Alignment Software (see Li H. and Durbin R. (2010) Fast and accurate long-read alignment with Burrows-Wheeler Transform. Bioinformatics.) using the merged reads by pear against the hg19 genome. Amplification statistics QC can be performed by analyzing one or more of, but not limited to, total reads, number of mapped reads, number of on-target mapped reads, and number of counted reads.

[0051] The methods described herein can include constructing a background error model using normal or healthy liquid samples, in exemplary embodiments, normal or healthy plasma samples, sequenced in the same sequencing run to account for run-specific artifacts. In some embodiments, 5, 10, 15, 20, 25, 30, 40, 50, 100, 150, 200, 250, or 250 or more normal or healthy liquid samples, in exemplary embodiments, plasma samples, are analyzed in the same sequencing run. The number of samples that can be sequenced in the same sequencing run can range from 5 to 500, 5 to 400, 5 to 300, 5 to 250, 20 to 250, 30 to 250, 50 to 250, 75 to 250, 100 to 250, 50 to 500, or 100 to 500. In exemplary embodiments, sample barcodes are used. In some exemplary embodiments, 20, 25, 40, or 50 normal samples (e.g., plasma samples) can be analyzed in the same sequencing run.To consider noise and contamination, outlier samples can be repeatedly removed from the model.In some embodiments, samples with Z-scores greater than 5, 6, 7, 8, 9, or 10 are removed from data analysis.For each base substitution at genomic locus, the DOR weighted average and the standard deviation of error can be calculated.

[0052] The method described herein can include determining the number of matched positions in the aligned DNA sequences, and calculating the identity percentage, which can be calculated by dividing the number of matched positions by the total number of aligned DNA sequences and multiplying by 100. A matched position refers to a position in the aligned DNA sequences where the same nucleotide occurs at the same position. The identity percentage over a specific length can be determined by counting the number of matched positions over that length, dividing this number by the length, and multiplying the resulting value by 100. A non-limiting example for calculating percent identity is if (i) a 500 nucleotide DNA target sequence is compared to a subject DNA sequence, (ii) an alignment program presents 200 nucleotides from the target DNA sequence aligned with a region of the subject DNA sequence, where the first and last nucleotides of that 200 nucleotide region match, and iii) the number of matches across those 200 aligned nucleotides is 180, then the 500 nucleotide nucleic acid target sequence is 200 in length and can have 90 percent sequence identity across its length (i.e., 180, 200 x 100 = 90).

[0053] In some embodiments, DOR uniformity can be measured using standard methods, such as, but not limited to, DOR slope, normalized median read depth (nmDOR), or read breadth (BOR). The DOR slope represents the slope of the line in the linear portion of a list of loci sorted in descending order of DOR. The closer to zero, the better, since it represents a flat line. In some embodiments, DOR uniformity can be measured using the percentage of reads falling within the 90th to 95th percentiles. For this measurement, loci are sorted in descending order of DOR. In an exemplary embodiment, a DOR distribution using the 90th to 95th percentiles contains 5 percent of the reads. The reads for all loci between the 90th and 95th percentiles can be counted and divided by the sum of the reads for all loci.

[0054] In some embodiments, the magnitude of the slope of the DOR can be less than 0.005, 0.001, 0.0005, 0.0001, 0.00005, 0.00001, 0.000005, or 0.000001. The magnitude of the slope of the DOR can be 0 to 0.005, e.g., 0.000001 to 0.005, e.g., 0.000005 to 0.00001, 0.00001 to 0.00005, 0.00005 to 0.0001, 0.0001 to 0.0005, 0.0005 to 0.001, or 0.001 to 0.005. The 90th to 95th percentile read percentage can be 0.2 to 9 percent, e.g., 0.2 to 8 percent, 0.2 to 7 percent, 0.2 to 6 percent, 0.4 to 9 percent, 0.4 to 8 percent, 0.4 to 7 percent, 0.4 to 6 percent, 1 to 9 percent, 1 to 8 percent, 1 to 7 percent, 1 to 6 percent, 2 to 9 percent, 2 to 8 percent, 2 to 7 percent, 2 to 6 percent, 3 to 9 percent, 3 to 8 percent, 3 to 7 percent, 3 to 6 percent, 0.2 to 1.0 percent, 1 to 2 percent, 2 to 3 percent, 2 to 4 percent, 3 to 4 percent, 4 to 5 percent, 5 to 6 percent, 6 to 8 percent, or 7 to 9 percent. In some embodiments of the methods described herein, the method or amplification step of the method can produce a composition comprising at least 100 different amplicons (e.g., at least 300, 500, 750, 1,000, 2,000, 5,000, 7,500, 10,000, 15,000, 19,000, 20,000, 25,000, 27,000, 28,000, 30,000, 40,000, 50,000, 75,000, or 100,000 non-identical amplicons), with the magnitude of the DOR slope in any of the ranges described herein or the percentage of reads within the 90-95th percentile in any of the ranges described herein.In some embodiments, the different amplicons can range from 100 to 500,000, 100 to 400,000, 100 to 300,000, 100 to 200,000, 100 to 100,000, 100 to 75,000, 100 to 50,000, 100 to 40,000, 100 to 30,000, 100 to 25,000, 100 to 20,000, or 100 to 15,000 non-identical amplicons.

[0055] In some embodiments of the methods described herein, in addition to, or in some embodiments as an alternative to, analyzing altered (increased or decreased) methylation levels in a sample, one or more other factors can be analyzed, if desired. These factors can be used to improve the accuracy of diagnosis or prognosis (such as determining the presence or absence of cancer or an elevated risk of cancer, classifying cancer, or staging cancer). These factors can also be used to select a particular therapy or treatment regimen that is likely to be effective in a subject.

[0056] Detection limit Representative exemplary methods described herein are for detecting target region amplicons generated by targeted amplification and / or selectively enriched, which are used to determine the methylation status of one or more methylation sites of interest, in exemplary embodiments, multiple methylation sites of interest, on DNA molecules in a DNA sample. A target region can contain one, two, three, four, five, six, or more CpG sites that are typically predominantly methylated or co-methylated with cytosine nucleotides in cells of a particular origin (e.g., cancer). For example, a target region can be all or part of a CpG island that is predominantly methylated in tumors. In exemplary embodiments, sample DNA molecules containing a target region are fragments of a subject's genomic DNA or circulating free DNA (cfDNA). Target regions can be selectively amplified and / or selectively enriched using the methods described herein. Representative methods described herein, in some embodiments, have detection limits as low as 1.0%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, or 0.001% (by mass), and the methods can detect fully methylated DNA molecules present at 1.0%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, or 0.001% or greater in a mixture of DNA molecules. In some embodiments, the methods described herein can distinguish between samples having 1.0%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, or 0.001% or greater (by mass) of artificial fully methylated DNA molecules and samples having 0% artificial fully methylated DNA molecules. Exemplary methods described herein, in some embodiments, have a low detection limit of 1.0%, 0.5%, 0.1%, 0.05%, 0.02%, 0.01%, 0.005%, 0.002%, or 0.001% differentially methylated allele fraction (DMAF), which estimates the fraction of differentially methylated alleles in circulating cell-free DNA across the target region.

[0057] In some embodiments, measurements can be adjusted for bias, such as bias due to differences in amplification efficiency, or adjusted for sequencing error. In some embodiments, the distinction between methylated and unmethylated samples can be analyzed using a fully methylated control sample, such as a fully methylated plasmid control (e.g., pUC19), for normalization of quantitative results. In some embodiments, the distinction between the methylated samples listed in this paragraph can be achieved after normalizing the detected and typically quantified signals using one or more (e.g., 2, 3, 4, 5, or 6) controls that do not have the MSRE recognition site.

[0058] In certain embodiments, ctDNA is detected when target region amplicon is detected.Because this target region amplicon is usually generated when MSRE recognition site on sample DNA molecule is methylated, and this methylation is known to occur in the promoter region of tumor suppressor gene in the early stage of carcinogenesis.In certain embodiments, this method can detect when ctDNA exists in 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 2.5%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, 0.002%, 0.001% or less of the circulating free DNA in sample. In some embodiments, the methods described herein detect or are capable of detecting ctDNA from a sample when present in a range between a lower limit of 0.1%, 0.05%, 0.02%, 0.01%, 0.005%, 0.002%, or 0.001% and an upper limit of 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 2.5%, 1%, or 0.5% of the total cfDNA amount in the sample. In some embodiments, the methods described herein detect or are capable of detecting circulating tumor DNA of 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, 0.002%, 0.001% or less as a percentage of total circulating free DNA (cfDNA) from the sample. The percentage of ctDNA in a cfDNA sample can be determined or approximated by the variant allele frequency (VAF) of one or more DNA mutations, such as single nucleotide variants ("SNVs") present in tumor cells but absent in normal cells (see, for example, WO2019200228, the entire contents of which are incorporated herein by reference). VAF can be used as a surrogate indicator of the percentage of ctDNA in a cfDNA sample. At least in colorectal cancer (CRC) patients, there is a strong correlation (approximately 0.9) between DMAF and VAF, regardless of clinicopathological characteristics such as stage, histological type, age, or gender. Methylation-based assays are promising tools for cancer detection and disease burden quantification.

[0059] Nucleic acid treatment before MSRE treatment Typically, the methods described herein include ligating a nucleic acid adapter to a sample DNA molecule, or in exemplary embodiments, to a nucleic acid derivative generated therefrom. In exemplary embodiments, the sample DNA molecule is from a subject. In some embodiments, ligating the nucleic acid adapter is performed after the sample DNA molecule has been fragmented to form fragmented DNA molecules. Typically, the method includes exposing the sample DNA molecule to one or more polymerases or kinases, such as Klenow Large Fragment Polymerase and T4 polynucleotide kinase (PNK), and a ligase, such as T4 ligase. In some embodiments, the sample DNA molecule or the fragmented DNA molecule is exposed to one or more polymerases and / or kinases to generate the nucleic acid derivative generated therefrom. In some embodiments, the method further includes ligating an adapter to the nucleic acid derivative generated therefrom.

[0060] Typically, in the exemplary methods described herein, adapters are ligated to sample DNA molecules. In exemplary embodiments, prior to such ligation, extracted or isolated DNA molecules can be modified to form sample nucleic acid derivatives, e.g., to make them more amenable to adapter ligation. For example, the sample DNA molecules can be blunt-ended, nucleotides can be added to the sample DNA molecules or their blunt-ended derivatives, and / or phosphate groups can be added or removed from the ends of the sample DNA molecules or their derivatives. In some embodiments, prior to ligation, the sample DNA molecules can be blunt-ended, and then a single adenosine base can be added to the 3' end. Prior to ligation, the DNA can be cleaved using a restriction enzyme or some other cleavage method. During ligation, the 3' adenosine of the sample fragment and the complementary 3' thymidine overhang of the adapter can increase ligation efficiency. Typically, in exemplary embodiments, adapter ligation is performed using T4 ligase.

[0061] In exemplary embodiments, adapters containing one or more universal priming sequences are utilized in the methods described herein. In some embodiments, the adapters are Y adapters, for example, in exemplary methods of sequencing target region amplicons using NGS. In some embodiments, each adapter comprises a universal priming site. Typically, the adapters do not comprise any MSRE recognition site for any MSRE used in the method.

[0062] In some embodiments, each adapter further comprises a sample barcode. Thus, multiple samples can be analyzed in the same sequencing reaction. The sample barcode can be used to process the data according to the sample from which it was generated.

[0063] In some embodiments, each adapter further comprises a molecular barcode. In some embodiments, the number of adapters with different molecular barcodes is 10 to 1,000, and the ratio of the total number of sample nucleic acid or cfDNA molecules in the ligation reaction to the number of different molecular barcodes is at least 1,000:1. In certain embodiments, the number of different molecular barcodes in the ligation reaction ranges from 10 to 50, 10 to 100, 50 to 200, 100 to 300, 200 to 500, 300 to 600, 500 to 700, 600 to 800, or 700 to 1,000. In some embodiments, the ligation reaction contains at least 1, 10, 20, 30, 40, 50, or at least 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 different molecular barcodes. In some embodiments, the ratio of the total number of sample nucleic acid or cfDNA molecules to the number of different molecular barcodes in ligation reaction is at least 10,000: 1. In some embodiments, the ratio of the total number of sample nucleic acid or cfDNA molecules to the number of different molecular barcodes in ligation reaction is 50,000: 1 to 50: 1, 25,000: 1 to 100: 1, 10,000: 1 to 100: 1, 10,000: 1 to 8,000: 1 to 500: 1, 5,000: 1 to 200: 1, or 10,000: 1 to 50: 1. In some embodiments, the methods disclosed herein result in at least 100, 200, 500, 750, 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000 different molecular barcodes per sample nucleic acid or cfDNA molecule.

[0064] Methylation-sensitive restriction enzymes (MSREs) The methods described herein typically involve contacting a sample DNA molecule or a nucleic acid derivative produced therefrom with one or more MSREs. MSREs are useful for analyzing the methylation status of cytosine residues at CpG sites. As the name suggests, these enzymes cannot cleave palindromic target sites if the cytosine residues are methylated. MSRE target sites range in size from 4 bp up to 76 bp, but typically range from 4 bp to 8 bp.

[0065] In certain embodiments, the methods described herein generally involve contacting a sample DNA molecule or a nucleic acid derivative generated therefrom with one or more MSREs. The MSREs selectively cleave the sample nucleic acid when the MSRE recognition site is unmethylated, but do not cleave when the MSRE recognition site is methylated. An "isoschizomer" of an MSRE is a restriction enzyme that recognizes the same recognition site as a methylation-sensitive restriction enzyme but cleaves both methylated and unmethylated CGs. An isoschizomer of a selected MSRE may be used in a control reaction. Non-limiting examples of methylation-sensitive restriction enzymes include AatII, Acc65I, AccI, AciI, AclI, AfeI, AgeI, AgeI-HF™, AhdI, AleI-v2, ApaI, and ApaLI. ApeKI, AscI, AsiSI, AvaI, AvaII, BaeI, BanI, BbvCI, BceAI, BcgI, BcoDI, BfuAI, BglI, BmgBI, BsaAI, BsaBI, BsaHI, BsaI-HF (trademark registered)v2, BseYI, BsiE, BsiWI, BsiWI-HF (trademark registered), BslI, BsmAI, Bs mBI-v2, BsmFI, BspDI, BspEI, BsrBI, BsrFI-v2, BssHII, BstAPI, BstBI, BstUI, BstZ17I-HF (trademark) (registered), BtgZI, Cac8I, ClaI, DpnI, DraIII-HF (registered trademark), DrdI, EaeI, EagI-HF (registered trademark), EarI, EciI, Eco 53kI, EcoRI, EcoRI-HF (trademark registered), EcoRV, EcoRV-HF (trademark registered), Esp3I, FauI, Fnu4HI, FokI, FseI, Fsp I, HaeII, HgaI, HhaI, HinP1I, HincII, HinfI, HpaI, HpaII, Hpy166II, Hpy188III, Hpy99I, HpyA V, HpyCH4IV, KasI, MboI, MluI, MluI-HF (trademark registered), MmeI, MspA1I, MwoI, NaeI, NarI, NciI, NgoMIV, NheI-HF (trademark registered), NlaIV, NotI, NotI-HF (trademark registered), NruI, NruI-HF (trademark registered), Nt.BbvCI, Nt.BsmAI, Nt.Examples of suitable MSREs include CviPII, PaeR7I, PaqCI, PleI, PluTI, PmeI, PmlI, PshAI, PspOMI, PspXI, PvuI, PvuI-HF™, RsaI, RsrII, SacI-HF™, SacII, SalI, SalI-HF™, Sau3AI, Sau96I, ScrFI, SfaNI, SfiI, SfoI, SgrAI, SmaI, SnaBI, SrfI, StyD4I, TfiI, TseI, TspMI, XhoI, XmaI, and / or ZraI, and thus, in some embodiments, one or more MSREs can comprise them. In an exemplary embodiment, one or more MSREs can comprise HhaI, HpaII, BstUI, and / or HpyCH4IV.

[0066] In any of the aspects and embodiments disclosed herein, one or more MSREs can be replaced with one or more methylation-dependent restriction enzymes (MDREs). MDREs selectively cleave sample nucleic acids when one or more nucleotides in the MDRE recognition site are methylated. Those skilled in the art will understand how to modify the methods provided herein to include an MDRE or to replace an element listed as an MSRE with an MDRE. Thus, in some embodiments, one or more MDREs can be used in place of an MSRE or in the absence of an MSRE. In some embodiments, one or more MDREs can be used in combination with one or more MSREs. In some embodiments, the one or more MDREs can be AbaSI, AoxI, BisI, BlsI, DpnI, FspEI, GlaI, GluI, KroI, LpnPI, MalI, MspJI, MteI, PcsI, PkrI, or SgeI.

[0067] MSREs (or MDREs) can be selected based on differentially methylated CpG sites in target DNA molecules, such as tumor or ctDNA from a specific cancer target, or a spectrum of diverse tumors. Additional criteria for selection include low background methylation in normal tissues, the size and number of cleavage fragments, the number of base pairs in the recognition sequence, whether cleavage results in blunt-ended or tailed-ended fragments, and whether the enzymes have the same or similar reaction conditions so that the contacting steps can be performed under the same set of conditions and / or in a single reaction.

[0068] In some embodiments, two or more MSREs (and / or MDREs), a plurality of MSREs (and / or MDREs), or a set of MSREs (and / or MDREs) can be used to contact a target DNA molecule containing one or more CpG sites of interest. The number of selected MSREs in certain embodiments is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20, 25, 50, or 100. In some embodiments, the number of MSREs is in the range of 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20, 25, 50, or 100, or 2 to 3, 4, 5, 6, 7, 8, 9, 10, or 20, 25, 50, or 100, or 5 to 6, 7, 8, 9, 10, or 20, 25, 50, or 100. In some embodiments, the number of MSREs ranges from 1 to 10, 1 to 5, 2 to 5, 3 to 5, 4 to 5, 3 to 7, 5 to 10, 6 to 10, or 7 to 10. Therefore, criteria such as target sites, methylation status of the target sites, number of base pairs in the tailed terminal fragments, buffer conditions, reaction conditions including, but not limited to, incubation time and temperature for optimal activity, and inactivation time and temperature for each MSRE can be used to select multiple MSREs and / or MDREs, or sets of MSREs and / or MDREs, for inclusion in the method. Because activity, measured in units, is specific to each enzyme, target DNA molecules can be contacted with 1 to 5 units (U) of each MSRE in the reaction sample. In some embodiments, target DNA molecules are contacted with 1 to 5 U, 1.5 to 4 U, 2 to 3 U, 2.5 to 4 U, or 3 to 5 U of each MSRE. In some embodiments, one or more of the MSREs are selected from HpaII, SalI, BbeI, NotI, SmaI, XmaI, MboI, BstUI, BstBI, ClaI, MluI, NaeI, NarI, PvuI, SacII, HpyCH41V, HhaI, and combinations thereof. In an exemplary embodiment, one or more MSREs are selected from one or more of HpaII, HhaI, HpyCH41V, and BstU1.In some embodiments, the one or more MSREs are selected from one or more of HpaII, HhaI, HpyCH41V, or BstU1. In some embodiments of the methods as described herein, the contacting comprises contacting with two or more MSREs. In some embodiments, the contacting comprises contacting with three or more MSREs. In some embodiments, the contacting comprises contacting with four or more MSREs. In some embodiments, the contacting comprises contacting with two or more MSREs in a single reaction.

[0069] In some embodiments, the cleavage buffer conditions include an MSRE and / or an MDRE capable of cleaving an MSRE site and / or an MDRE site under the same cleavage buffer conditions. In some embodiments, the cleavage buffer conditions include 5 to 500 mM potassium acetate, for example, 25 to 100 mM potassium acetate. In some embodiments, the cleavage buffer conditions include 2 to 200 mM Tris-acetate, for example, 10 to 40 mM Tris-acetate. In some embodiments, the cleavage buffer conditions include 1 to 100 mM magnesium acetate, for example, 5 to 20 mM magnesium acetate. In some embodiments, the cleavage buffer conditions include 10 to 1000 μg / ml recombinant albumin, for example, 50 to 200 μg / ml recombinant albumin. In some embodiments, the pH is 6.9 to 8.9 at 25°C, e.g., 7.4 to 8.4, 7.5 to 8.3, 7.6 to 8.2, 7.7 to 8.1, or 7.8 to 8, or about 7.9 at 25°C. In some embodiments, the cleavage buffer conditions include 1 to 100 mM Bis-Tris-propane-HCl, e.g., 5 to 20 mM Bis-Tris-propane-HCl. In some embodiments, the cleavage buffer conditions include 1 to 100 mM MgCl2, e.g., 5 to 20 mM MgCl2. In some embodiments, the pH is 6 to 8 at 25°C, e.g., 6.5 to 7.5, 6.6 to 7.4, 6.7 to 7.3, 6.8 to 7.2, or 6.9 to 7.1, or about 7.0 at 25°C. In some embodiments, the cleavage buffer conditions comprise 5 to 500 mM NaCl, for example, 25 to 100 mM NaCl. In some embodiments, the cleavage buffer conditions comprise 1 to 100 mM Tris-HCl, for example, 5 to 20 mM Tris-HCl. In some embodiments, the cleavage buffer conditions comprise 10 to 1000 mM NaCl, for example, 50 to 200 mM NaCl. In some embodiments, the cleavage buffer conditions comprise 5 to 500 mM Tris-HCl, for example, 25 to 100 mM Tris-HCl.In some embodiments, the cleavage buffer conditions comprise 25-100 mM potassium acetate, 10-40 mM Tris-acetate, 5-20 mM magnesium acetate, and 50-200 μg / ml recombinant albumin, and the pH is 7.6-8.2 at 25° C. In some embodiments, the cleavage buffer conditions comprise 5-20 mM Bis-Tris-propane-HCl, 5-20 mM MgCl2, and 50-200 μg / ml recombinant albumin, and the pH is 6.7-7.3 at 25° C. In some embodiments, the cleavage buffer conditions comprise 25-100 mM NaCl, 5-20 mM Tris-HCl, 5-20 mM MgCl2, and 50-200 μg / ml recombinant albumin, and the pH is 7.6-8.2 at 25° C. In some embodiments, the cleavage buffer conditions comprise 50-200 mM NaCl, 25-100 mM Tris-HCl, 5-20 mM MgCl2, and 50-200 μg / ml recombinant albumin, with a pH of 7.6-8.2 at 25°C.

[0070] amplification The method in some aspects described herein comprises carrying out one or, in some embodiments, two or more amplifications.In certain exemplary embodiments, such amplification comprises at least one targeted amplification, in which at least one primer used for amplification, in certain embodiments, both primers of a primer pair, one or more primer pairs, or one primer pair, is designed to bind to a specific nucleic acid sequence (i.e., a target-specific primer) at or near the genomic region of interest, usually within the genomic region, to generate a target region amplicon.In some embodiments, the method described herein comprises one or more universal amplifications.

[0071] Many amplification techniques can be used with the methods described herein. For example, such amplification can be isothermal amplification (e.g., recombinase polymerase amplification (RPA) (Kersting et al. 2014 Microchim Acta 181(13-14), 1715-1723, (incorporated by reference in its entirety)), ligase-based amplification, PCR, or a combination thereof (e.g., ligation-mediated PCR). In some exemplary embodiments, the targeted amplification is targeted PCR(s) performed using a PCR reaction mixture containing a primer pair, or in exemplary embodiments, a set of primer pairs, and MSRE-exposed adapted DNA molecules (e.g., MSRE-exposed adapted cfDNA), or universally amplified MSRE-exposed adapted DNA molecules generated therefrom.

[0072] Figure 4 illustrates various potential locations of primer binding sites on a DNA molecule subjected to a representative exemplary method described herein. In the example shown in Figure 4, the adapted DNA molecule includes a sample DNA region flanked by two Y adapters, each with a 5' adapter strand (350) and a 3' adapter strand (355). The sample DNA region in this example includes a target region (shown with light gray diagonal lines) with two CpG sites within the target region (shown as circled "mC") and one CpG site near the target region, all of which are methylated in this example. Typically, at least one primer of a primer pair used for targeted amplification described herein is a target-specific primer designed to bind to a specific nucleic acid sequence within or near, typically within, the genomic region of interest. In illustrative examples, this can be a genomic region where epigenetic changes, such as changes in DNA methylation, are associated with or indicative of the formation or presence of cancer, e.g., promoter regions of tumor suppressor genes, and in some embodiments, DNA methylation markers for specific cancer types. The target-specific primers (320, 380, 325, 385) can be designed to bind to any sequence within or near the target region for amplification of the target region or a portion of the target region. In some embodiments, the target-specific primers can be designed to bind to a sequence containing a CpG site covered by an MSRE recognition sequence. In other embodiments, the target-specific primers can be designed to bind to a sequence upstream or downstream of one or more CpG sites covered by one or more MSRE recognition sequences. One advantage of the methods described herein is increased flexibility in primer / probe design for targeted amplification or enrichment. In some embodiments, one primer or set of primer pairs among one or more primer pairs in the reaction mixture used for targeted amplification is a universal primer (310, 315) that binds to a primer binding site on at least one of the adapters.Thus, for example, in such an embodiment, a universal primer (310) that binds to an adapter primer binding site can be used for an amplification reaction along with a target-specific primer (325 or 385) that binds to a primer binding site on the sample DNA region.

[0073] The target-specific primers typically define the ends of a target region amplicon, which typically encompasses at least a portion of the target region. In some embodiments, PCR can be performed using two target-specific primers 320, 385. The target region amplicon in such embodiments will extend from the sample DNA region bound by the target-specific primer 320 on the 5' end to the sample DNA region bound by the primer 385 on the 3' end. In some embodiments, PCR can be performed using a universal primer 310 and a target-specific primer 385. The target region amplicon in such embodiments will extend from the sample DNA region bound by the target-specific primer 385 on the 3' end of one strand to the end of the sample DNA fragment on the 5' end of that strand.

[0074] In exemplary embodiments, target-specific primers can be designed to generate a target region amplicon that encompasses one or more CpG sites in the target region that are covered by one or more MSRE recognition sequences. The one or more MSRE recognition sites in such embodiments can be located within or near the intended target region amplicon, or can overlap the target-specific primer binding site.

[0075] In some embodiments, sample DNA fragments may contain MSRE sites located outside the intended target region amplicon (e.g., MSRE recognition site 370 associated with the intended target region amplicon to be generated using primers 320, 385). In such embodiments, universal preamplification of adaptor-attached DNA molecules can selectively enrich for sample DNA fragments in which all MSRE recognition sites are methylated, including MSRE sites outside the amplicon, such as 370. Sample DNA fragments in which one or more MSRE sites outside the amplicon are unmethylated are typically cleaved by one or more MSREs and typically are not amplified using universal primers that bind to priming sites on the adaptors, even if all MSRE sites within the intended target region amplicon are methylated. In cancer cells, CpG sites within CpG islands in regulatory regions are predominantly co-methylated. Therefore, the methods disclosed herein not only increase flexibility in primer or probe placement / design, but also improve specificity in detecting DNA from cancer cells.

[0076] In some embodiments, both primers in at least one of the one or more primer pairs, or both primers in a set of primer pairs, are target-specific primers. Thus, for example, primer 320 can be used in combination with primer 325 or primer 385. In some embodiments, both primers in more than one of the one or more primer pairs, or both primers in each primer pair in a set of primer pairs, are target-specific primers. Such target-specific primer binding sites, in some embodiments, can overlap a target CpG site covered with one or more MSRE recognition sites.

[0077] As discussed above, in some methods described herein, universal amplification can be performed before targeted amplification. Such universal amplification can be performed, for example, using a universal primer pair (310, 315) that binds to the primer binding site of the adapter. Such universal amplification (e.g., universal PCR) can be included in certain exemplary embodiments in which the sample DNA molecule is expected to include one or more MSRE recognition sites located outside the intended target region amplicon, as discussed above. Thus, in some embodiments, the methods described herein include performing universal PCR using a plurality of adapted DNA molecules or a plurality of adapted cfDNAs and a universal PCR primer pair including a primer designed to bind to the universal primer binding sequence on the adapter to generate amplified and MSRE-exposed adapted DNA molecules or amplified and MSRE-exposed adapted cfDNAs before performing one or more targeted PCRs or sets of targeted PCRs using the amplified and MSRE-exposed adapted DNA molecules or MSRE-exposed adapted cfDNAs.

[0078] In some embodiments, at least one primer in at least one primer pair, or a plurality of primer pairs, or each primer pair in one or more primer pairs, or each primer pair in a set of primer pairs is a target-specific primer.The method described herein can comprise a primer pair that is designed to bind to uncleaved version of the adaptor-attached DNA molecule or adaptor-attached cfDNA, but not to uncleaved adaptor-attached DNA molecule.

[0079] In some embodiments of any aspect described herein, one or more of the target regions, or set of target regions, comprises one or more MSRE recognition sites that overlap a primer binding site of one or more primer binding sites, or a primer binding site of a set of primer binding sites.

[0080] In exemplary embodiments, the one or more primer pairs are a set of primer pairs. In some embodiments, the set of primer pairs is a set of 2 to 1000, 2 to 500, 2 to 250, 2 to 200, 2 to 150, 2 to 100, 2 to 50, or 2 to 10 primer pairs, or a set of 5 to 1000, 5 to 500, 5 to 250, 5 to 200, 5 to 150, 5 to 100, 5 to 50, or 5 to 10 primer pairs, or a set of 50 to 250 or 100 to 200 primer pairs.

[0081] In some embodiments, at least one of the primer pairs comprises a universal primer and a target-specific primer. In some embodiments, at least one of the primer pairs comprises two target-specific primers. In some embodiments, at least one of the primers comprises a sequencing tag. In some embodiments, at least one of the primers comprises a sample index. In some embodiments, at least one of the primers comprises a biotin modification. In some embodiments, performing PCR further comprises using a primer comprising a sequencing tag. In some embodiments, performing PCR further comprises using a primer comprising a sample index. In some embodiments, the primers of the primer pair are probe-dependent primers, and the amplification is target capture polymerase chain reaction. In some embodiments, the target regions each comprise a set of two or more MSRE recognition sites that are differentially methylated in one or more cancers. In some embodiments, the set of loci comprises two or more loci that are differentially methylated in different types of cancer. In some embodiments, the target regions each comprise three or more MSRE recognition sites that are differentially methylated in cancer. In some embodiments, the MSRE recognition sites in one group of target regions are methylated in one type of cancer, and the MSRE recognition sites in another group of target regions are methylated in a different type of cancer.

[0082] Some of the adapted DNA molecules, in exemplary embodiments, some of the adapted DNA molecules exposed to an MSRE, comprise one or more target regions, and at least a portion of the one or more target regions comprise one or more MSRE recognition sites. In some embodiments, after the step of contacting the adapted DNA molecules with one or more MSREs, and in exemplary embodiments, further after the step of universal amplification, the methods described herein further comprise performing PCR using the plurality of adapted DNA molecules exposed to an MSRE, or the plurality of pre-amplified adapted DNA molecules exposed to an MSRE, and one or more primer pairs designed to amplify the one or more target regions, or portions thereof, in the adapted DNA molecules exposed to an MSRE, or copies thereof, and a target region amplicon is generated if all of the MSRE recognition sites within or near the intended target region amplicon are methylated.

[0083] In some embodiments, the methods described herein can further include using one or more primer pairs designed to bind to primer binding sites on the adapters of the adapted DNA molecule, thereby amplifying the uncleaved adapted DNA molecule or a copy thereof exposed to the MSRE. In some embodiments, the adapted DNA molecule or a copy thereof exposed to the MSRE contains one or more MSRE recognition sites outside the intended target region amplicon, and a target region amplicon is generated when all of the MSRE recognition sites in the adapted DNA molecule exposed to the MSRE, including one or more MSRE recognition sites outside the intended target region amplicon, are methylated. In some embodiments of the methods that include using one or more primer pairs designed to bind to primer binding sites on the adapters of the adapted DNA molecule exposed to the MSRE, the one or more primer pairs are designed to bind to the primer binding site on only one of the adapters for each adapted DNA molecule exposed to the MSRE, thereby amplifying the adapted DNA molecule or a copy thereof exposed to the MSRE.

[0084] In some embodiments, in the methods described herein, PCR is performed using one or more primer pairs designed to amplify one or more target regions in the adapted DNA molecule exposed to the MSRE, thereby generating target region amplicons. In some embodiments, both primers of a plurality of one or more primer pairs, or both primers of a set of primer pairs, are target-specific primers. In some embodiments, each primer of one or more primer pairs, or each primer of a set of primer pairs, is a target-specific primer. In some embodiments, both primers of at least one primer pair of one or more primer pairs, or both primers of a set of primer pairs, are target-specific primers.

[0085] In some embodiments, in the methods described herein, PCR is performed using one or more primer pairs designed to bind to a primer binding site on an adapter of an adapted DNA molecule exposed to an MSRE, thereby amplifying uncleaved adapted DNA molecules exposed to an MSRE. In some embodiments, at least one primer of at least one primer pair of one or more primer pairs, or at least one primer of a set of primer pairs, is designed to bind to a primer binding site on one of the adapters. In some embodiments, at least one primer of multiple primer pairs of one or more primer pairs, or at least one primer of a set of primer pairs, is designed to bind to a primer binding site on one of the adapters. In some embodiments, at least one primer of each primer pair of one or more primer pairs, or at least one primer of each primer pair of a set of primer pairs, is designed to bind to a primer binding site on one of the adapters. In some embodiments, at least one of the primer binding sites of a primer pair can comprise one or more MSRE sites. In some embodiments, one of the primer binding sites of a primer pair can comprise one or more MSRE sites. In some embodiments, both primer binding sites of a primer pair can comprise one or more MSRE sites. In some embodiments, neither primer binding site of the primer pair contains an MSRE site. In some embodiments, the amplicon generated by performing PCR using the primer pair contains one or more MSRE sites, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 MSRE sites. In some embodiments, the amplicon generated by performing PCR using the primer pair does not contain an MSRE site.In some embodiments, the one or more MSRE sites are within 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides of the primer binding site. In some embodiments, one or more of the one or more MSRE sites overlaps with the primer binding site.

[0086] In some embodiments, one or both primer binding sites of a primer pair can comprise at least a portion of one of the adapter sequences. In some embodiments, one primer binding site of a primer pair can comprise at least a portion of an adapter sequence. In some embodiments, both primer binding sites of a primer pair can comprise at least a portion of an adapter sequence. In some embodiments, neither primer binding site of a primer pair comprises either of the adapter sequences.

[0087] In some embodiments, the multiple adapted DNA molecules having one or more MSRE recognition sites, or the adapted cfDNA having one or more MSRE recognition sites, each comprise one or more target regions, each comprising two or more MSRE recognition sites. In some embodiments, the intended target region amplicon comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 MSRE sites. In some embodiments, the intended target region amplicon comprises 1 to 20 MSRE sites, for example, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 MSRE sites, or 2 to 15, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 MSRE sites.

[0088] Methods as described herein can, in some embodiments, include multiple amplification cycles (e.g., multiple PCR temperature cycles), and in some embodiments, can include several consecutive PCR reactions performed during the same set of temperature cycles. In some embodiments, the amplification cycles can include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 cycles. In some embodiments, the amplification cycles can include at least 7, 8, 9, or 10 cycles. In exemplary embodiments, the amplification cycles can include at least 11, 12, 13, 14, 15, 16, or 17 cycles.

[0089] Typically, in the embodiments described herein, PCR amplification is carried out by adding a PCR reaction mixture to a DNA template (e.g., adapted sample DNA or cfDNA exposed to MSRE), followed by adding a polymerase enzyme, and then amplifying through multiple amplification cycles. In some embodiments, the PCR reaction mixture contains one or more primer pairs, deoxynucleotides (dNTPs), a PCR reaction buffer, and deionized water. In some embodiments, the dNTPs can include a mixture of dATP, dCTP, dGTP, and dTTP. In some embodiments, the final concentration of each dNTP in the reaction mixture can be in the range of 0.05 mM to 0.5 mM dNTP, e.g., 0.05 mM to 0.5 mM, 0.05 mM to 0.1 mM, 0.05 mM to 0.15 mM, 0.05 mM to 0.2 mM, 0.05 mM to 0.25 mM, 0.05 mM to 0.3 mM, 0.05 mM to 0.35 mM, 0.05 mM to 0.4 mM, 0.05 mM to 0.45 mM, 0.1 mM to 0.5 mM, 0.15 mM to 0.5 mM, or 0.2 mM to 0.5 mM, 0.25 mM to 0.5 mM, 0.3 mM to 0.5 mM, 0.35 mM to 0.5 mM, 0.4 mM In exemplary embodiments, the final concentration of each dNTP in the reaction mixture is 0.15 mM to 0.25 mM. In some embodiments, the final concentration of each dNTP in the reaction mixture is 0.2 mM.

[0090] PCR buffer solutions create an environment suitable for polymerase chain reaction and can contain many different components, including magnesium chloride (MgCl), potassium chloride (KCl), dimethyl sulfoxide (DMSO), and glycerin or bovine serum albumin (BSA). In some embodiments, the concentration of KCl can be 25-50 mM, 25-75 mM, 25-100 mM, 30-100 mM, 50-100 mM, or 70-100 mM. In some embodiments, the MgCl concentration can be 0.5 mM to 5 mM, 0.5 mM to 4.5 mM, 0.5 mM to 4.0 mM, 0.5 mM to 3.5 mM, 0.5 mM to 3.0 mM, 0.5 mM to 2.5 mM, 0.5 mM to 2.0 mM, 0.5 mM to 1.5 mM, 1.0 mM to 5 mM, 1.5 mM to 5 mM, 2.0 mM to 5 mM, 2.5 mM to 5 mM, 3.0 mM to 5 mM, 3.5 mM to 5 mM, 4.0 mM to 5 mM, or 4.5 mM to 5 mM. In some embodiments, the MgCl concentration is 2.0 mM.

[0091] In exemplary embodiments, the buffer solution is Q5™ Reaction Buffer (B9027S, New England Biolabs, Inc.). In some embodiments, the reaction buffer is Standard Taq Reaction Buffer (B9014S, New England Biolabs, Inc.). In some embodiments, the reaction buffer is Standard Taq (Mg-free) Reaction Buffer (B9015S, New England Biolabs, Inc.).

[0092] In some embodiments, a DNA polymerase is used to generate DNA amplicons using DNA as a template. In some embodiments, the polymerase is a Q5® DNA polymerase, such as Q5® High-Fidelity DNA Polymerase (M0491S, New England BioLabs, Inc.) or Q5® Hot Start High-Fidelity DNA Polymerase (M0493S, New England BioLabs, Inc.). Q5® High-Fidelity DNA polymerase is a high-fidelity, thermostable DNA polymerase with 3' to 5' exonuclease activity fused to a processivity-enhancing Sso7d domain. Q5® High-Fidelity DNA polymerase lacks 5' to 3' exonuclease activity and strand displacement activity.

[0093] In some embodiments, the polymerase is T4 DNA polymerase (M0203S, New England BioLabs, Inc.). T4 DNA polymerase catalyzes the synthesis of DNA in the 5' to 3' direction and requires the presence of a template and a primer. This enzyme has a 3' to 5' exonuclease activity that is significantly more active than that found in DNA Polymerase I. T4 DNA polymerase lacks 5' to 3' exonuclease activity and strand displacement activity.

[0094] In some embodiments of any of the aspects described herein, the length of the primer can be 10 to 100 nucleotides, e.g., 10 to 75 nucleotides, 10 to 40 nucleotides, 10 to 35 nucleotides, 10 to 30 nucleotides, 10 to 20 nucleotides, 15 to 100 nucleotides, 20 to 100 nucleotides, 25 to 100 nucleotides, 30 to 100 nucleotides, 35 to 100 nucleotides, 40 to 100 nucleotides, 45 to 100 nucleotides, 50 to 100 nucleotides, 55 to 100 nucleotides, 60 to 100 nucleotides, 65 to 100 nucleotides, 70 to 100 nucleotides, or 75 to 100 nucleotides. In some embodiments, the length of the primer ranges from 5 to 50 nucleotides, e.g., 5 to 40 nucleotides, 5 to 20 nucleotides, or 5 to 10 nucleotides. In some embodiments, the primers are 5 to 50 bp, 10 to 40 bp, 15 to 30 bp, 15 to 25 bp, 20 to 40 bp, 25 to 50 bp, or 30 to 50 bp in length, 25 to 100 bp, 35 to 100 bp, 45 to 100 bp, 55 to 100 bp, 65 to 100 bp, or 75 to 100 bp in length.

[0095] In some embodiments of any of the aspects or embodiments described herein, the number of primer pairs can range from 1 to 100,000, each primer pair binding to one or more primer binding sequences. In some embodiments, the primer pair is part of a set of primer pairs. In some embodiments, the set of primers ranges from 2 to 100,000, 2 to 10,000, 2 to 1,000, 2 to 100, 2 to 50, 10 to 100, 50 to 100, 100 to 200, 100 to 500, 100 to 1,000, 100 to 10,000, 100 to 100,000, 1,000 to 100,00, or 10,000 to 100,000 primer pairs. In some embodiments, the number of primer pairs can range from 10 to 10,000, 10 to 1,000, 10 to 100, 10 to 50, 10 to 40, 10 to 30, 15 to 30, or 15 to 25 primer pairs.

[0096] In some embodiments, PCR is used to generate extremely short amplicons. cfDNA (for example, fetal cfDNA in maternal serum or cancer cfDNA released by necrosis or apoptosis) is highly fragmented. In the case of fetal cfDNA, fragment sizes are distributed in a Gaussian fashion, with an average of 160 bp, a standard deviation of 15 bp, a minimum size of about 100 bp, and a maximum size of about 220 bp. The methylation site(s) of interest may occupy any position from the beginning to the end of various fragments originating from a specific locus. Because cfDNA fragments are short, the likelihood that both primer sites are present, that is, the likelihood that a fragment of length L contains both forward and reverse primer sites, is the ratio of the length of the amplicon to the length of the fragment. Under ideal conditions, assays with amplicons of 45, 50, 55, 60, 65, or 70 bp will successfully amplify from 72%, 69%, 66%, 63%, 59%, or 56% of available template fragment molecules, respectively. Thus, in some embodiments, the target amplicon length generated by the methods described herein is 40-100, 40-75, or 45-70 bp. In certain embodiments, most preferably related to cfDNA from samples of individuals suspected of having cancer, cfDNA is amplified using primers that result in a maximum amplicon length of 85, 80, 75, or 70 bp, and in certain preferred embodiments, 75 bp, and have a melting temperature of 50-65°C, and in certain preferred embodiments, 54-60.5°C. The amplicon length is the distance between the 5' ends of the forward and reverse priming sites. Amplicon lengths shorter than those typically used by those known in the art may result in more efficient measurement of desired methylation sites by requiring only short sequence reads. In one embodiment, a significant portion of the amplicons are between the lower end of the range of 25 bp and the upper end of the range of 100 bp, 90 bp, 80 bp, 70 bp, 65 bp, 60 bp, 55 bp, 50 bp, or 45 bp.

[0097] Probe-dependent primers In some embodiments, one or more primers described herein are probe-dependent primers. Probe-dependent primers (PDPs) have been disclosed (Pel, et al., "Rapid and highly-specific generation of targeted DNA sequencing libraries enabled by linking capture probes with universal primers," PLoS ONE 13(12):e0208283 (2018); WO 2020 / 039261, "Linked target capture and ligation," incorporated herein by reference in its entirety). Such embodiments can be considered LTC methods. Briefly, in LTC methods, PDPs are designed to incorporate a non-extendible capture probe linked 5'-5' to the primer. As discussed below, multiple linker types are possible. Typically, PDP probes are 30-70 nucleotides in length and contain or comprise a 3' inverted dT base to inhibit polymerase extension. In some embodiments, the probes are designed to cover the desired region with zero gap between the forward and reverse probes. In some embodiments, the probes are 20 to 100 nucleotides in length. In some embodiments, the probe size can be 20 to 40 nucleotides, 30 to 50 nucleotides, 40 to 60 nucleotides, 50 to 70 nucleotides, 60 to 80 nucleotides, 70 to 90 nucleotides, 80 to 100 nucleotides, 90 to 110 nucleotides, or 100 to 120 nucleotides in length. In some embodiments, at least one of the probes in a PDP pair includes a sample index.

[0098] In PDP, forward and reverse probes are designed to bind to nucleic acid sequences within or near a genomic region of interest on a sample DNA molecule, allowing enrichment of nucleic acid molecules containing the genomic region of interest or copies thereof. In some embodiments, at least one of the probe binding regions can contain one or more MSRE sites. In some embodiments, one of the probe binding regions can contain one or more MSRE sites. In some embodiments, both of the probe binding sites in a probe binding region can contain one or more MSRE sites. In some embodiments, neither of the probe binding sites in a probe binding region contains an MSRE site.

[0099] Typically, the primer portion of a PDP is a universal primer designed to bind to the universal primer site on the ligated adapter. In some embodiments, the PDP is designed with a sequencer binding sequence, such as an Illumina flow cell binding sequence, incorporated therein. In some embodiments, the sequencer flow cell binding sequence is located between the probe and the universal primer and adjacent to the primer. The ligated primers of the present invention may contain a sequencing tag to ensure that all cluster reads originate from the same ligated template molecule. The length of the primer can be extended or shortened at the 5' or 3' end to generate primers with a desired melting temperature. The annealing position of each primer pair can also be designed so that the sequence and length of the primer pair result in the desired melting temperature. In an exemplary embodiment, the primer is a universal primer with a low melting temperature that is complementary to a portion of the ligated adapter.

[0100] Primers can be tailed or untailed depending on specific needs. In some embodiments, the universal primer comprises an A tail. In some embodiments, the universal primer is blunt-ended. The length of the PDP primer can range from 5 to 40 nucleotides in length. In certain embodiments, the PDP primer is 10 to 25 nucleotides in length. In embodiments, the PDP primer can range from 5 to 15 nucleotides, 10 to 25 nucleotides, 15 to 35 nucleotides, or 25 to 40 nucleotides in length.

[0101] Typically, a probe-dependent primer includes a linker between the probe and the primer. The probe and primer portions of a PDP are typically linked by a polyethylene glycol derivative, an oligosaccharide, a lipid, a carbohydrate, a polymer, or a protein. In some embodiments, the linker is a PEG molecule or a derivative thereof. In some embodiments, the linker is an oligosaccharide. In some embodiments, the linker is a lipid. In some embodiments, the linker is a carbohydrate. In some embodiments, the linker is a polymer. In some embodiments, the linker is a protein or a portion thereof.

[0102] Hybrid Capture As shown in FIG. 1 , in some embodiments, one or more selective enrichment steps (152) can be used to enrich for DNA molecules containing one or more target regions. For example, the selective enrichment technique can involve fragment capture by hybridization (i.e., hybrid capture). While any hybrid capture method can be used to perform the methods described herein that involve a selective enrichment step, in some embodiments, the disclosed methods can involve using any of the hybrid capture methods disclosed herein to selectively enrich for DNA, e.g., cfDNA. Such selective enrichment steps can be performed after an amplification step, typically after a universal preamplification step, and sometimes immediately after the universal preamplification step. Furthermore, one or more selective enrichment steps can occur before one or more downstream universal or targeted amplification steps. For example, the methods described herein can include performing universal amplification to amplify uncleaved, adaptor-tagged cfDNA exposed to at least a plurality of MSREs to generate amplicons, followed by hybrid capture using hybrid capture probes to selectively enrich the amplified DNA molecules containing one or more target regions. As another example, the methods described herein can include performing targeted amplification to amplify one or more target regions from uncleaved adapted cfDNA molecules or copies thereof that have been exposed to a plurality of MSREs, where if the one or more target regions are present in the uncleaved adapted cfDNA molecules that have been exposed to the MSREs, target region amplicons are formed, and then performing a hybrid capture method using hybrid capture probes to selectively enrich for the one or more target region amplicons.

[0103] In one aspect, provided herein is a method for preparing a DNA molecule, and in an exemplary embodiment, a cfDNA molecule, useful for determining the methylation status of a selected region of a DNA molecule, comprising: a) ligating adapters to cfDNA obtained or derived from a first liquid sample, thereby forming a plurality of adapted cfDNAs, the adapted cfDNAs comprising adapted cfDNAs having one or more MSRE recognition sites; b) contacting the plurality of adapted cfDNAs with one or more MSREs, thereby generating MSRE-exposed adapted cfDNAs, wherein the plurality of MSRE-exposed adapted cfDNAs are not cleaved by the one or more MSREs, thereby forming MSRE-exposed uncleaved adapted cfDNAs; c) amplifying at least some of the uncut adaptored cfDNA exposed to the MSRE; d) selectively enriching a subset of the amplified, MSRE-exposed, uncleaved, adapted cfDNA having one or more target regions using a set of hybrid capture probes, each target region containing one or more MSRE recognition sites, and each probe of the set is designed to hybridize to one target region; e) quantifying the amount of at least some of the enriched subsets by performing a next generation sequencing reaction on the clonally amplified enriched subsets, or amplicons derived therefrom.

[0104] Hybridization capture utilizes a hybrid capture oligonucleotide probe complementary to a single strand of a specific target DNA sequence in a sample or DNA derived therefrom. In exemplary embodiments, the specific target DNA sequence overlaps or is found within a target region of a sample DNA molecule, such as cfDNA. Thus, when used in the methods described herein, a hybrid capture probe can be designed to bind to a DNA molecule containing at least one target region or a portion thereof. In some embodiments, the hybrid capture probe can be designed to bind to a target DNA sequence within or overlapping a target region, which in exemplary embodiments contains one or more MSRE recognition sites. In other examples, the hybrid capture probe can be designed to bind to a common region adjacent to but not overlapping the target region, which can be a common region added to some, most, nearly all, or all of the DNA in the sample, or a common region added to all amplicons using a common sequence on at least one primer of a primer pair. In exemplary embodiments, a hybrid capture probe or set thereof is designed to bind to a target DNA sequence within a target region or set of target regions, respectively.

[0105] The hybrid capture probe may be added to the prepared sample and hybridized through a denaturation-reannealing process to form a duplex of exogenous-endogenous fragments (e.g., a hybrid capture probe bound to a sample DNA molecule or DNA derived therefrom). These duplexes may then be physically separated from the sample by various means. In some embodiments, once the hybrid capture probe is removed, the sample DNA molecule or DNA derived therefrom can be amplified. Some methods for physically removing hybrid capture probes include covalently binding the hybrid capture probe to a solid support, such as a magnetic bead or chip. Another method for physically removing hybrid capture probes is by covalently binding them to a molecular moiety with a strong affinity for another molecular moiety. An example of such a molecular pair is biotin and streptavidin, such as those used in SURE SELECT (Agilent). Therefore, for example, a hybrid capture probe that binds to a target DNA sequence that overlaps or is within a target region of a DNA molecule derived from or obtained from a sample can be covalently attached with a biotin molecule, and after hybridization with the sample DNA or the DNA derived from the sample, the biotinylated hybrid capture probe can be pulled down using a solid support with immobilized streptavidin, and these hybridized with the DNA molecule derived from or obtained from the sample that contains the target region that includes the target DNA sequence recognized by the hybrid capture probe.Therefore, in some embodiments, the hybrid capture probe is directly or indirectly immobilized on a solid support.In some embodiments, the hybrid capture probe comprises a binding partner, such as biotin.

[0106] In some embodiments of any of the aspects described herein, the hybrid capture probe can be part of a set of at least two hybrid capture probes, each designed to bind to a different target sequence in the target region. In some embodiments, the set includes at least one hybrid capture probe per target region. In some embodiments, the set includes two or more hybrid capture probes per target region.

[0107] In some embodiments of any of the aspects described herein, the hybrid capture probe is 30 to 170 bases, 30 to 160 bases, 30 to 150 bases, 30 to 140 bases, 30 to 130 bases, 30 to 120 bases, 30 to 110 bases, 30 to 100 bases, 30 to 90 bases, 30 to 80 bases, 30 to 70 bases, 30 to 60 bases, 30 to 50 bases, 0 bases, 40 bases to 160 bases, 40 bases to 150 bases, 40 bases to 140 bases, 40 bases to 130 bases, 40 bases to 120 bases, 40 bases to 110 bases, 40 bases to 100 bases, 40 bases to 90 bases, 40 bases to 80 bases, 40 bases to 70 bases, 40 bases to 60 bases, 50 bases to 150 bases, 50 bases to 140 bases, 50 bases to 130 bases, 50 bases to 120 bases, 50 bases to 110 bases, 50 bases ~100 bases, 50 bases to 90 bases, 50 bases to 80 bases, 50 bases to 70 bases, 60 bases to 140 bases, 60 bases to 130 bases, 60 bases to 120 bases, 60 bases to 110 bases, 60 bases to 100 bases, 60 bases to 90 bases, 60 bases to 80 bases, 70 bases to 130 bases, 70 bases to 120 bases, 70 bases to 110 bases, 70 bases to 100 bases, 70 bases to 90 bases, 80 bases to 120 bases, 80 bases The length can range from 1 to 110 bases, 80 to 100 bases, 90 to 120 bases, 90 to 110 bases, 100 to 165 bases, 100 to 150 bases, 100 to 140 bases, 100 to 130 bases, 100 to 120 bases, 110 to 150 bases, 110 to 140 bases, 110 to 130 bases, 120 to 150 bases, or 130 to 160 bases.

[0108] When using hybrid capture upstream of a next-generation sequencing reaction, one way to increase the number of reads interrogating a position of interest is to shorten the length of the hybrid capture probe, as long as it does not result in a bias in the underlying enriched allele. The length of the hybrid capture probe must be long enough so that two hybrid capture probes designed to bind to two different target DNA sequences within the same target region hybridize with approximately equal affinity to the target sequence. In certain embodiments, using shorter probes results in a higher likelihood that the hybrid capture probe will bind to DNA molecule fragments from a liquid sample, such as cfDNA. Furthermore, in some embodiments, the hybrid capture probe can be designed to bind to two DNA sequences on the target DNA within the same target region that are separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11-20, or 20 or more nucleotides. In some embodiments, for each target region, hybrid capture probes can be designed to bind to different DNA sequences within the overlapping target region by 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 110, 120, or 5-120 nucleotides. Thus, hybrid capture can be used to selectively enrich for DNA molecules containing the targeted region in a DNA sample.

[0109] Comparison of two or more samples In some embodiments, the methods described herein can include comparing two or more samples. In some embodiments, the methods described herein can include comparing three, four, five, six, seven, eight, nine, ten, or more samples. In some embodiments, the methods described herein can include comparing two samples, and such methods further include performing the method on a second sample, and determining the methylation status of the set of target regions in the first sample further includes comparing the amount of target region amplicons derived from the first sample with the amount of target region amplicons derived from the second sample. In some embodiments, the second sample is from a second subject. In some embodiments, the second sample is derived from a cell line. In some embodiments, the method is performed on the first and second samples simultaneously. In some embodiments, a method as described herein can include comparing two samples, and such a method further includes performing the method on a second sample, and determining the methylation status of the set of target DNA molecules in the first sample further includes comparing the amount of amplified methylated target DNA molecules from each of the set of target DNA molecules in the first sample with the amount of amplified methylated target DNA molecules from each of the set of target DNA molecules in the second sample. In some embodiments, the first subject is suspected of having or at risk for a disease, and the second subject is not suspected of or at risk for a disease. In some embodiments, the second sample is from the first subject. In some embodiments, the second sample is taken at a first timepoint and the first sample is taken at a second timepoint. In some embodiments, the first timepoint precedes the second timepoint. In some embodiments, determining includes comparing the amount quantified for the target region amplicon derived from the first sample with the amount quantified for the target region amplicon derived from the second sample using the same method.In some embodiments, the method further includes, in addition to a first performance of the method, a second performance of the method performed simultaneously with or at a different time from the first performance, wherein the second performance does not include a contacting step, and the method further includes comparing the amount quantified in the first performance with the amount quantified in the second performance to determine the methylation state of the target nucleic acid. In some embodiments, the determining includes comparing the amount quantified for the target region amplicon to a predetermined threshold amount.

[0110] Representative Embodiments In this representative embodiment section, non-limiting exemplary aspects and embodiments are provided herein and are further discussed throughout the specification. For the sake of brevity and convenience, not all aspects and embodiments disclosed herein, nor all possible combinations of the disclosed aspects and embodiments, are listed in this section. Additional embodiments and aspects are provided in other sections of the specification. Furthermore, it will be understood that embodiments are provided that are specific embodiments for many aspects and can be combined with any other embodiment or aspect, for example, as discussed throughout this disclosure. Given the full disclosure set forth herein, it is intended that any individual embodiment listed below or in this full disclosure can be combined with any aspect listed below or in this full disclosure. This may be the case in the case of additional elements that can be added to an aspect, or in the case of narrower elements relative to elements already present in an aspect. Such combinations may be provided as non-limiting representative combinations and / or may be more specifically discussed in other sections of this detailed description.

[0111] One embodiment provided herein is a method for preparing a deoxyribonucleic acid (DNA) molecule useful for determining the methylation status of a genomic region of interest, the method comprising: a) ligating adaptors to sample DNA molecules obtained or derived from a first sample from a first subject, thereby forming a plurality of adapted DNA molecules, including adapted DNA molecules having a methylation-sensitive restriction enzyme (MSRE) recognition site; b) contacting a plurality of adapted DNA molecules with one or more MSREs, thereby generating MSRE-exposed adapted DNA molecules, wherein adapted DNA molecules exposed to at least a first plurality of MSREs having one or more unmethylated MSRE recognition sites are cleaved by at least one of the MSREs, and adapted DNA molecules exposed to a second plurality of MSREs are not cleaved by any of the MSREs, thereby forming MSRE-exposed uncleaved adapted DNA molecules; c) performing one or more amplification reactions to amplify one or more target regions from the uncleaved adapted DNA molecules or copies thereof exposed to the MSRE, wherein if the one or more target regions are present in the uncleaved adapted DNA molecules exposed to the MSRE, a target region amplicon is formed.

[0112] In some embodiments, each intended target region amplicon has one or more MSRE recognition sites. In some embodiments, each intended target region amplicon has two or more MSRE recognition sites. In some embodiments, at least 10% of the intended target region amplicons each contain at least two MSRE recognition sites. In some embodiments, at least 15%, 20%, 25%, 30%, or 35% of the intended target region amplicons each contain at least two MSRE recognition sites. In some embodiments, the methods described herein can include at least one intended target region amplicon containing at least three, four, five, or six MSRE recognition sites. In some embodiments, the methods described herein can include at least one intended target region amplicon containing between 2 and 45 MSRE recognition sites.

[0113] In some embodiments, the sample DNA molecules are circulating free DNA (cfDNA). In some embodiments, one or more target region amplicons are generated by one or more polymerase chain reactions (PCR) using one or more primer pairs, and in exemplary embodiments, at least one primer in each of the one or more primer pairs is a target-specific primer. In some embodiments, one or more target region amplicons are generated using one or more capture probes designed to hybridize to one or more target regions. In some embodiments, one or more target region amplicons are generated by one or more ligation target capture (LTC) reactions using one or more probe-dependent primer pairs. In some embodiments, the method further comprises detecting one or more target region amplicons. In some embodiments, the one or more target regions are a set of target regions, and in certain embodiments, the one or more primer pairs are a set of primer pairs, each configured to amplify one target region of the set. In some embodiments, the method further comprises quantifying the amount of at least one of the one or more target region amplicons.

[0114] In some embodiments, performing the one or more amplifications includes performing universal amplification, wherein uncleaved adaptor-attached cfDNA exposed to at least a plurality of MSREs is amplified to generate amplicons, and the method further includes selectively enriching one or more target regions from the amplicons using one or more hybrid capture probes to generate enriched DNA molecules containing the one or more target regions. In some embodiments, the method further includes detecting the one or more enriched DNA molecules containing the one or more target regions. In some embodiments, the one or more target regions are a set of target regions, and the one or more hybrid capture probes are a set of hybrid capture probes, each configured to selectively bind to the sequence of one target region of the set. In some embodiments, the set of hybrid capture probes includes one or more probes capable of binding to two or more target regions of the set. In some embodiments, the method further includes quantifying the amount of enriched DNA molecules containing at least one of the one or more target regions.

[0115] In another aspect, provided herein is a method for preparing a DNA molecule useful for determining the methylation status of a selected region of a DNA molecule, the method comprising: a) ligating adapters to circulating free DNA (cfDNA) obtained or derived from a first liquid sample, thereby forming a plurality of adapted cfDNAs, including adapted cfDNAs having MSRE recognition sites; b) contacting the plurality of adapted cfDNAs with one or more MSREs, thereby generating MSRE-exposed adapted cfDNAs, wherein the plurality of MSRE-exposed adapted cfDNAs are not cleaved by the one or more MSREs, thereby forming MSRE-exposed uncleaved adapted cfDNAs; c) performing a first amplification step in which at least a plurality of the uncleaved adaptor-attached cfDNAs exposed to the MSRE are amplified; d) performing a second amplification step to amplify a set of target regions from the amplified and MSRE-exposed uncleaved adapted cfDNA, each target region having one or more MSRE recognition sites, wherein a target region of the set is amplified if the target region is present in one or more of the amplified and MSRE-exposed uncleaved adapted cfDNA, thereby generating a target region amplicon; e) quantifying the amount of at least some of the set of target region amplicons by performing a next generation sequencing reaction on the clonally amplified target region amplicons or amplicons derived therefrom.

[0116] In some embodiments, the first amplification step comprises one or more PCRs using universal primers. In some embodiments, the second amplification step comprises one or more PCRs using a set of primer pairs, each pair of the set designed to amplify a target region having one or more MSRE recognition sites, and at least one primer of each primer pair is a target-specific primer. In some embodiments, the second amplification step comprises one or more LTC reactions using a set of probe-dependent primer pairs, each pair of the set designed to amplify a target region having one or more MSRE recognition sites.

[0117] In one embodiment, provided herein is a method for preparing deoxyribonucleic acid (DNA) containing a methylation-sensitive restriction enzyme (MSRE) recognition site, the method comprising: a) ligating an adaptor to sample DNA obtained or derived from a first sample from a first subject, thereby forming an adapted DNA molecule, the adapted DNA molecule comprising an adapted DNA molecule having a methylation-sensitive restriction enzyme (MSRE) recognition site; b) contacting the adapted DNA molecule with one or more MSREs, thereby generating an MSRE-exposed adapted DNA molecule, wherein the adapted DNA molecule exposed to the multiple MSREs having an unmethylated MSRE recognition site is cleaved by at least one of the MSREs; c) performing one or more targeted polymerase chain reactions (PCRs) using a PCR reaction mixture comprising adapted DNA molecules exposed to an MSRE and one or more primer pairs designed to amplify one or more target regions in the adapted DNA molecules exposed to the MSRE, wherein each target region comprises one or more MSRE recognition sites, and one or more of the one or more target regions are amplified if all of the MSRE recognition sites in the adapted DNA molecules exposed to the MSRE that make up the one or more target regions are methylated.

[0118] In another aspect, provided herein is a method for preparing a DNA molecule useful for determining the methylation status of a selected region of a DNA molecule, the method comprising: a) ligating adapters to circulating free DNA (cfDNA) obtained or derived from a first liquid sample, thereby forming a plurality of adapted cfDNAs, including adapted cfDNAs having MSRE recognition sites; b) contacting the plurality of adapted cfDNAs with one or more MSREs, thereby generating MSRE-exposed adapted cfDNAs, wherein the plurality of MSRE-exposed adapted cfDNAs are not cleaved by the one or more MSREs, thereby forming MSRE-exposed uncleaved adapted cfDNAs; c) amplifying at least some of the uncut adaptored cfDNA exposed to the MSRE; d) selectively enriching a subset of the amplified, MSRE-exposed, uncleaved, adapted cfDNA having one or more target regions using a set of hybrid capture probes, each target region containing one or more MSRE recognition sites, and each probe of the set is designed to hybridize to one target region; e) quantifying the amount of at least some of the enriched subsets by performing a next generation sequencing reaction on the clonally amplified enriched subsets, or amplicons derived therefrom.

[0119] In some embodiments, the methods disclosed herein further comprise determining the methylation status of one or more target regions. In some embodiments, determining comprises comparing the amount quantified for the target region amplicon derived from the first sample with a preset value. In some embodiments, determining comprises comparing the amount quantified for the target region amplicon derived from the first sample with the amount quantified for the target region amplicon derived from a second sample using the same method.

[0120] In some embodiments, the amplification comprises one or more rounds of PCR using universal primers. In some embodiments, the one or more target regions is a set of target regions.

[0121] While embodiments of the present disclosure are amenable to various modifications and alternative forms, specific embodiments have been shown by way of example in the drawings and are described in detail below. The intention, however, is not to limit the disclosure to the particular embodiments described. On the contrary, the disclosure is intended to cover all modifications, equivalents, and alternatives falling within the scope of the present disclosure as defined by the appended claims.

[0122] The following non-limiting examples are provided purely for purposes of illustrating exemplary embodiments and in no way limit the scope and spirit of the present disclosure. Furthermore, any invention disclosed or claimed herein should be understood to encompass all variations, combinations, and permutations of any one or more features described herein. One or more features may be explicitly excluded from the scope of a claim, even if a specific exclusion is not explicitly set forth herein. It should also be understood that the disclosure of a reagent for use in a method is intended to be synonymous with (and support) that method, unless one of ordinary skill in the art understands otherwise. Additionally, where the specification and / or claims disclose a method, any one or more of the reagents disclosed herein may be used in that method, unless one of ordinary skill in the art understands otherwise. [Example]

[0123] Example 1. Comparison of MSRE digestion before and after adapter ligation This example demonstrates a comparison of two workflows for determining the methylation status of target DNA molecules in cfDNA samples from healthy donors and CRC patients. The workflows differ in the order of steps in which MSRE is used to digest sample DNA molecules relative to the adapter ligation step in library preparation, as shown in Figures 5A and 6 (Workflow 1) and 5B and 7 (Workflow 2), respectively.

[0124] It was hypothesized that workflow 1 (MSRE digestion before adapter ligation to DNA molecules from the sample) would avoid loss of information / reads through unexpected digestion from MSRE sites outside the intended amplicon. However, MSRE digestion before adapter ligation would likely result in increased background noise and inefficient preamplification, biasing the generation of amplicons from shorter digested fragments that typically lack methylated targets. On the other hand, given that CpG sites in certain regions of cancer cells are known to be predominantly comethylated, the potential loss of information / reads due to workflow 2 (MSRE digestion after adapter ligation) could be minimized or even ignored with appropriate target profiling. Furthermore, MSRE digestion after adapter ligation may increase assay specificity and allow greater flexibility in the placement / design of target-specific primers, because adapter-attached DNA molecules containing unmethylated off-amplicon MSRE sites would be cleaved by MSRE and would not be amplified in the universal amplification step.

[0125] As shown in Figure 5A, the first workflow (Workflow 1) involves digesting a DNA sample with a mixture of MSREs before any library preparation. As shown in Figure 5B, the second workflow (Workflow 2) involves digesting a DNA sample with a mixture of MSREs after adapter ligation. The DNA reactions and products of the workflows in Figures 5A and 5B are shown in Figures 6 and 7, respectively. In both workflows, an MSRE cocktail / mixture of HpaII, HhaI, BstU1, and HpyCH4IV, or a control solution without any MSREs, was used in the digestion steps of the experiments described herein. MSREs were selected based on the MSRE recognition sites in the proposed target region amplicon.

[0126] Unless otherwise noted, DNA samples containing (i) 100% methylated artificial human gDNA, (ii) 100% unmethylated artificial human gDNA, (iii) cfDNA from healthy subjects, or (iv) cfDNA from CRC patients were used for the experiments described herein (411A, 411B). In some experiments, samples were spiked with 0.01% unmethylated lambda phage and 0.0006% methylated pUC19 DNA by mass as a control and to normalize results. Human gDNA and lambda + pUC19 DNA samples were sheared and size-selected using an R230 sonicator and AFA-TUBE TPX plates (Covaris) to mimic the size of cfDNA prior to use.

[0127] In workflow 1, the DNA sample (411A) was then treated with the MSRE cocktail or a control solution without MSRE (430A, 520A). A solution containing 30 ng of DNA in a 40 μl volume was added to an MSRE cocktail containing 0.125 μl each of the following MSREs (NEB): 20 U / μl HpaII, 20 U / μl HhaI, 10 U / μl HpyCH4IV, and 10 U / μl BstUI in 4.5 μl of 10X CutSmart Buffer (NEB), or to a control solution containing 4.5 μl of 10X CutSmart Buffer (NEB) in 0.5 μl of water alone. Digestion was carried out at 37°C for 1 hour and 60°C for 30 minutes, followed by inactivation at 80°C for 20 minutes. The sample was purified with 3 volumes of Ampure XP beads (Beckman Coulter) to retain potential small fragments and eluted with 40 μl of DSB-Tween® 20 elution buffer.

[0128] In workflow 1, the treated DNA samples were then blunt-ended, A-tailed, and ligated with adapters that did not contain an MSRE site or were fully methylated. The ligated DNA samples were then universally amplified (420A, 530A). Specifically, the DNA samples were blunt-end repaired using Klenow Large Fragment Polymerase and T4 polynucleotide kinase (PNK) (Thermofisher) and A-tailed using Klenow Fragment exonuclease (3'-5' exo) (Thermofisher). Nucleic acid adapters that did not contain an internal MSRE site or were fully methylated were ligated using T4 ligase (Thermofisher), and the 5' ends were phosphorylated using PNK, resulting in Y-adapted fragments (530A, Figure 6). The MSRE-treated, adapter-attached DNA molecules were purified using the Kingfisher AMPure XP-Purification protocol and eluted in Tris buffer solution. Library amplification was performed by PCR set for 20 cycles in a Veriti Thermocycler (Applied Biosystems) using a universal primer designed to bind to the primer binding site on the adapter on the Y-adapted DNA molecules and Kapa HiFi Polymerase (Kapa Biosystems), resulting in amplified adapted DNA molecules (552A in Figure 6).

[0129] In Workflow 2, the individual step protocols were the same as in Workflow 1, except that the DNA sample (411B) was blunt-end repaired and A-tailed, and then adapters were ligated as described to generate Y-adapted DNA samples (420B, 520B) prior to the MSRE digestion step (430B, 530B). The MSRE-treated, adapted DNA sample was then universally amplified (440B) as described above, resulting in amplified adapted DNA molecules (552B in Figure 7).

[0130] In both workflows, universal amplification was followed by targeted amplification using target-specific primer pairs (450A, 450B, 550A, 550B). Specifically, targeted PCR reactions were performed to amplify target regions within the universally amplified, adaptor-tagged DNA molecules using approximately 220 primer pairs designed to amplify regions known to be hypermethylated in CRC patients. To maximize primer selection while avoiding primer interactions, primers that interact with each other were separated into two pools: Pool 1 and Pool 2.

[0131] PCR reaction mixtures were prepared using pooled target-specific primer pairs (558A / B, 562A / B). PCR reactions were performed on a Veriti PCR machine with an initial denaturation step at 98°C for 2 minutes, followed by 14 cycles of the following steps: 95°C for 30 seconds, 62.5°C for 15 minutes, and 72°C for either 30 seconds or 1 minute, followed by a final extension at 72°C for 2 minutes. The resulting amplified DNA molecule (554A / B) contained the target region or a portion of it.

[0132] Six microliters of each targeted PCR reaction well was pooled and frozen. For sequencing, the pooled samples were purified using a QiAquick PCR purification column (Qiagen) and finally eluted in 50 μl of elution buffer. The purified pooled samples were quantified using qPCR and run in triplicate on an Agilent BioAnalyzer DNA High-Sens assay at a 1:20 dilution.

[0133] Sequencing (560A / B) was performed at 2x50bp. A total of 56 samples were analyzed, with a read depth of over 100K for human targets. Data were analyzed focusing on the enrichment of methylated target DNA molecules and the depletion of unmethylated target DNA molecules in genomic DNA samples. Additionally, the performance of the two workflows was compared for cfDNA from healthy donors and CRC patients. Other key targets, including on-target rate, uniformity, and dropout rate, were also evaluated.

[0134] Enzyme efficiency evaluation using lambda controls The efficiency of MSRE enzymes was first calculated based on the digestion of unmethylated lambda DNA using Workflow 1 (W1). The efficiency of BstU1, HhaI, and HpyCH4IV was found to be approximately 99.5%, while the efficiency of HpaII was approximately 75% (data not shown).

[0135] The efficiency of the MSRE enzymes was further evaluated based on the digestion of unmethylated lambda DNA using both Workflow 1 and Workflow 2. Unmethylated lambda DNA samples were treated with either the MSRE cocktail or a control solution without any MSREs. Data for lambda targets containing one or more MSRE recognition sites were extracted and analyzed separately for each MSRE in the cocktail. For each enzyme, the depth of read (DOR) of amplicons containing one or more recognition sites for the enzyme was normalized to the median DOR of the methylated pUC19 control and analyzed and compared between Workflow 1 (W1) and Workflow 2 (W2), as well as between treated (MSRE) and control samples.

[0136] As can be seen in Figure 8, the normalized DOR for samples processed without any MSRE (Control.W1 and Control.W2) was approximately 4 for W1 and approximately 6 for W2. Samples processed with the MSRE cocktail (MSRE.W1 and MSRE.W2) yielded similar results for BstUI, HhaI, and HpyCH4IV, with normalized DORs approaching zero for both workflows, whereas for HpaII, the measured normalized DOR was approximately 1.5 for W1 and approximately 1 for W2, thus corresponding to the efficiency of each restriction enzyme as determined in prior experiments. Overall, these results demonstrate that the unmethylated lambda control is sensitive to MSRE digestion and that both Workflow 1 and Workflow 2 perform similarly on the unmethylated lambda control sample. Evaluation of enzyme efficiency and workflow specificity using artificially 100% methylated and 0% methylated human gDNA samples

[0137] Next, the efficiency of the MSRE enzyme in Workflow 1 and Workflow 2 was determined using artificial human gDNA. Human gDNA samples that were artificially 100% or 0% methylated were processed using Workflow 1 or Workflow 2 with the MSRE cocktail or a control solution without any MSRE. Using Workflow 1 and Workflow 2, depth of read (DOR) was calculated for the following samples: a 100% methylated gDNA sample processed with the MSRE cocktail, a 100% methylated gDNA sample processed with the control cocktail (no MSRE), a 0% methylated gDNA sample processed with the MSRE cocktail, and a 0% methylated gDNA sample processed with the control cocktail. Data for MSRE targets containing one or more cleavage sites were extracted and analyzed separately for each MSRE in the cocktail. For each enzyme, the depth of read (DOR) of amplicons containing one or more recognition sites for the enzyme was normalized to the median DOR of the methylated pUC19 control and analyzed and compared between workflow 1 (W1) and workflow 2 (W2), and between treated (MSRE) and control samples.

[0138] Results for each of the targeting primer pools are shown. Figure 9 (Pool 1) and Figure 10 (Pool 2) show a comparison of the normalized DOR readouts for two pools of human targets per MSRE recognition site between 100% and 0% methylated human gDNA samples treated with either the control mix or the MSRE mix using W1 or W2. As expected, HpaII demonstrated lower efficiency, primarily using the targeting primers from Pool 1 (Figure 9, HpaII results). Overall, however, for both targeting primer pools, MSRE digestion before library preparation (W1, Figure 5A) and after ligation (W2, Figure 5B) showed sufficient cleavage in the unmethylated samples (the last two columns of each graph in Figure 9 and the last two columns of each graph in Figure 10, labeled unmethylated W1-MSRE and unmethylated W2-MSRE), but was preserved (undigested) in the 100% methylated samples (the third and fourth columns of each graph in Figure 9 and the third and fourth columns of each graph in Figure 10, labeled 100% methylated W1-MSRE and 100% methylated W2-MSRE). These results therefore demonstrate that unmethylated artificial human gDNA is sensitive to MSRE treatment, and that both workflows were sufficient to distinguish between methylated and unmethylated human gDNA.

[0139] Comparison of DOR between cfDNA samples from CRC patients and healthy donors using workflow 1 and workflow 2 To assess whether workflows 1 and 2 could be used to distinguish between signals from healthy donors and CRC patients, we evaluated their individual performance on human amplicons on cfDNA samples from healthy donors and cfDNA samples from CRC patients using pooled target-specific primer pairs designed to amplify regions known to be hypermethylated in CRC patients.

[0140] First, cfDNA samples from CRC patients and healthy donors were analyzed using the workflow 1 protocol. All assays were normalized using non-MSRE sites. Figure 11 shows a heatmap of the normalized DOR of targets in pool 1 from samples from three individual CRC patients, C1, C2, and C3 (columns 3, 4, and 5, respectively), compared with three healthy individuals, H1, H2, and H3 (columns 6, 7, and 8). The VAFs for the C1, C2, and C3 samples were 25.31%, 20.52%, and 3.18%, respectively. Controls of 100% methylated and unmethylated human gDNA are shown in columns 1 and 2, respectively. Using Primer Pool 1 and the Workflow 1 protocol, high VAF samples C1 (25.31%) and C2 (20.52%) showed clear DOR enrichment compared to healthy donor samples with targets from Primer Pool 1, while low VAF sample C3 had a DOR comparable to that of healthy donor samples. The background of healthy donors was not as clean as unmethylated gDNA.

[0141] Next, high-VAF cfDNA samples from CRC patients and healthy controls were analyzed using either the Workflow 1 (W1) or Workflow 2 (W2) protocols, and the results of the two pools were combined. All assays were normalized to the pUC19 median. Figure 12 shows a heatmap of the normalized and combined DORs of Pool 1 targets and Pool 2 targets from two individual CRC patient samples (C1 and C2) compared with three and two healthy controls using Workflow 1 (W1) or Workflow 2 (W2). The VAFs for the C1 and C2 samples were 25.31% and 20.52%, respectively. Both W1 and W2 showed good discrimination between the high-VAF samples C1 (25.31%) and C2 (20.52%) and the healthy cfDNA samples. However, W2 had a much cleaner background of the healthy cfDNA samples compared to W1.

[0142] Next, cfDNA samples from CRC patients with low VAF and healthy controls were analyzed using either Workflow 1 (W1) or Workflow 2 (W2) protocols. All assays were normalized to the pUC19 median. In Figure 13, the left panel shows a heatmap of the normalized DOR of targets from pool 1 from three individual CRC patient samples, C3, C4, and C5, compared to healthy controls using either the W1 or W2 protocol. The right panel shows a heatmap of the normalized DOR of targets from pool 2 from C4 and C5 samples compared to healthy controls using the W2 protocol. The VAFs for the C3, C4, and C5 samples were 3.19%, 2.51%, and 1.78%, respectively. As can be seen in Figure 13, W2 provides a much cleaner background of healthy control cfDNA and better resolution in the low VAF CRC samples compared to W1. This is a key advantage of Workflow 2. This is because cfDNA samples for cancer screening or diagnostic assays, including early cancer screening assays, typically have low VAF of ctDNA, e.g., the average VAF for stage II and III CRC samples is less than 0.1%.

[0143] To further test the detection limit of Workflow 2, we measured the analytical resolution of Workflow 2 using Pool 1 targets and the Workflow 2 protocol, as discussed above, with 1%, 0.5%, 0.1%, 0.05%, and 0% (by mass) fully methylated artificial human gDNA. DOR was normalized to the median DOR of human non-MSRE controls. Samples containing 1%, 0.5%, 0.1%, or 0.05% fully methylated gDNA could be distinguished from samples containing 0% fully methylated gDNA.

[0144] Additional cfDNA samples from 13 healthy subjects and 13 CRC patients (11 with a VAF <0.1% and 2 with a VAF of 5%) were also tested using the workflow 2 protocol, as discussed above. The DORs for all samples were normalized to the median values ​​for non-MSRE sites in humans. As can be seen in Figure 14A (pool 1) and Figure 14B (pool 2), most CRC cfDNA samples with a VAF <0.05% exhibited distinct patterns compared to cfDNA samples from healthy donors. Targets in primer pool 2 demonstrated better resolution than primer pool 1 in low VAF samples, particularly in the 0.09%–0.05% VAF range. This suggests that the analytical resolution of workflow 2 can be further improved by target profiling and selection and / or further protocol optimization.

[0145] Example 2. Clinical performance of Workflow 2 To evaluate the clinical performance of the MSRE approach and demonstrate its potential for use in early cancer detection and recurrence monitoring, we developed a methylation-based classifier using machine learning algorithms to distinguish between patients with colorectal cancer (CRC), those in remission, and healthy individuals. Initial evaluation was performed by comparing the global methylation profiles of CRC and normal samples (tissue and blood) available in The Cancer Genome Atlas (TCGA) database and the Gene Expression Omnibus (GEO) dataset. We identified <800 CRC-specific CpG targets for initial evaluation. To develop and test the classification model, we analyzed 50 ctDNA-positive CRC patients (24% stage I, 40% stage II, 24% stage III, and 12% stage IV), 10 ctDNA-negative patients (CRC patients in remission), and 36 healthy controls.

[0146] Workflow 2 was utilized as discussed above (see Figure 5B and Figure 7). Using a machine learning model, we evaluated the best-performing CpG targets from less than 800 potential targets that effectively discriminated between CRC ctDNA-positive patients and healthy individuals. The median variant allele frequency (VAF) of single nucleotide variants (SNVs) in CRC ctDNA-positive samples was approximately 0.1%, with the majority of samples in the 0.01-1% range and few samples with a VAF between 1 and 5%. Using the best-performing CpG targets, observed methylation levels correlated with the VAF of SNVs detected by ctDNA testing (R2: 0.8). For example, Figure 15 demonstrates that using the top 40 targets, higher DOR is associated with higher SNV VAF.

[0147] Distinguishing CRC from normal individuals To evaluate whether the MSRE Workflow 2 approach could distinguish CRC ctDNA-positive samples from healthy or ctDNA-negative samples, MSRE-processed libraries were prepared as described above from 50 CRC ctDNA-positive samples, 10 CRC ctDNA-negative samples, and 36 healthy donor samples. 196 CRC-specific CpG regions were assayed in two pools (pool 1 and pool 2) using pooled target-specific primer sets (98 sets per pool) and sequenced using mPCR. Similar on-target rates were observed among the CRC-positive, CRC-negative, and healthy donor samples.

[0148] Assay DOR was normalized using total read counts, as shown in Figure 16A (pool 1) and Figure 16B (pool 2), and then compared across different sample groups. The solid line represents the mean normalized DOR, and the shaded area represents ± standard error. Targets were sorted based on the mean DOR ratio in CRC samples / healthy control samples on the x-axis. A subset of targets exhibited higher CRC / healthy control DOR ratios, with 53 / 196 assays having a CRC / healthy control DOR ratio greater than 10. Classification of CRC and healthy controls using random forests

[0149] To generate a model for classifying healthy and CRC samples, samples were divided into a 60% training set (57 samples) and a 40% test set (39 samples). The training set was used to generate a random forest model to distinguish CRC-positive from healthy samples.

[0150] Methylation targets were ranked by the Gini importance of random forest features, which calculates the importance of each feature as the sum of the number of divisions. Using the top targets, CRC samples with SNV VAF >0.05% demonstrated better separation than healthy control samples. The majority of the top target assays were able to provide differentiation between CRC and healthy control samples with VAF between 0.01 and 0.05% (Figure 17A and Figure 17B).

[0151] As shown in the heatmaps (Figures 18A and 18B), across the top targets, samples with a VAF above 0.05% can be distinguished from negative samples. Within each sample group, some sample-to-sample variability was observed, which may be due to the methylation and SNV signals not being perfectly correlated.

[0152] MSRE W2 Performance To evaluate the performance of MSRE Workflow 2 using the random forest model approach, the training set was used to generate random forest models using either all targets (Figure 19A) or a subset of targets (Figure 19B-D). As shown in Table 1 and Figure 19C, the top 20 targets in Pool 1 provided the highest sensitivity and specificity among the generated models, with a specificity of 86% and a sensitivity of 94% observed from a test set of 39 samples. Across all generated models, AUCs ranging from 0.92 to 0.95 were observed when classifying healthy samples from CRC samples. This is a probabilistic interpretation of the AUC.

[0153] At a specificity of 90%, a maximum sensitivity of approximately 95% was observed using the top 20 targets from pool 1 (Figure 19C). [Table 1]

[0154] MSRE Target Assay Review To understand the characteristics of targeted assays that could provide a clearer distinction between CRC and healthy controls, we examined the correlation between the mean CRC / healthy control DOR ratio and the number of cleavage sites. It was determined that assays with more MSRE cleavage sites provided a clearer distinction (higher CRC / healthy control DOR ratio, Figure 20).

[0155] Example 3. Hybrid capture of MSRE-treated libraries (MSRE+HC) Compared to targeted amplification approaches, targeted capture enrichment potentially assesses additional information beyond methylation levels observed using DOR, including fragment-level information that may enhance MSRE-based cancer detection. This example utilized 96 samples that underwent library preparation and MSRE processing as described in Example 2 above, followed by enrichment using a hybrid capture panel. As shown in the schematic diagram in Figure 21, the MSRE-processed libraries underwent amplification (440°C) followed by barcoding PCR (460°C) to add sample barcodes and P5 / P7 index sequences. Briefly, 200 ng of the MSRE-processed and amplified libraries were barcoded using barcoding primers to add P5 / P7 index sequences. The PCR reaction consisted of an initial denaturation step at 98°C for 3 minutes, followed by five cycles of 98°C for 20 seconds, 55°C for 20 minutes, and 68°C for 1 minute, followed by a final extension at 68°C for 5 minutes. Barcoded libraries were normalized and pooled (470°C) prior to hybrid capture (480°C) using probes specific for a target panel of up to 1482 probes. Up to 12 samples were pooled per hybrid capture reaction. Following the hybridization reaction, post-capture PCR was performed using P5 / P7 primers. The PCR reaction consisted of an initial denaturation step at 98°C for 45 seconds, followed by approximately 14 cycles (depending on panel size and number of pooled samples) of 98°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, followed by a final extension at 72°C for 1 minute. The post-capture libraries were then purified and sequenced using paired-end 2X 150bp sequencing on a dual-indexed Novaseq SP flow cell.

[0156] Target coverage between CRC-positive and normal samples To evaluate whether the MSRE+HC workflow can distinguish signals from normal individuals (healthy individuals or CRC ctDNA-negative individuals) from CRC ctDNA-positive individuals, libraries generated from MSRE-processed normal and CRC-positive samples were captured using the MSRE HC panel, as discussed above. The HC panel was designed to capture 860 CRC-specific CpG regions understood to be hypermethylated in CRC patients. These regions were identified by comparing the methylation landscape of CRC with adjacent normal or healthy tissue / blood samples available in The Cancer Genome Atlas (TCGA) database and public datasets on the Gene Expression Omnibus (GEO) repository. The panel also included 17 human non-MSRE targets. 855 / 860 target regions contained MSRE recognition sites, and the majority of target regions (±160 bp of the CpG) contained multiple MSRE recognition sites. 261 / 855 target regions contained recognition sites for all four MSREs, BstUI / HhaI / HpaII / HpyCH4IV, with each region having 4-38 cleavage sites, for a median of 13. Most target regions (481 / 855) contained recognition sites for three MSREs, BstUI / HhaI / HpaII, with each region having 3-42 cleavage sites, for a median of 13.

[0157] Coverage at CpG targets was calculated after deduplication. The DOR of the target assays was normalized to the DOR of the non-MSRE control, and then comparisons were performed for different sample groups. The normalized coverage at CpG targets for CRC ctDNA positive (CRC_POS), CRC ctDNA negative (NEG), and healthy samples is shown in Figure 22. Target assays were sorted based on the average DOR ratio in CRC / healthy samples on the x-axis. A subset of targets showed higher CRC / healthy DOR ratios, with 178 / 855 assays having a CRC / healthy DOR ratio greater than 10.

[0158] When using the top 20 and top 40 targets (ranked by CRC / healthy control DOR ratio), the majority of the top target assays can differentiate between CRC samples (VAF between 0.01% and 5%) and healthy control samples (Figure 23). As shown in the heatmaps, across the top 20 targets (Figure 24A) and top 40 targets (Figure 24B), CRC-positive samples (including low VAF samples) can be differentiated from CRC-negative samples and healthy control samples. Within each sample group, there is some variability from sample to sample.

[0159] MSRE+HC performance To evaluate the performance of the MSRE+HC workflow using a random forest model approach, samples were divided into a 60% training set and a 40% test set, and the training set was used to generate a random forest model using the top 40 high DOR ratio targets. As shown in Table 2 and Figure 25A, when using the MSRE+HC workflow, a sensitivity of 95% and a specificity of 100% were observed from a test set of 39 samples. The AUC observed when classifying healthy samples as CRC samples was 0.99. This is a probabilistic interpretation of the AUC.

[0160] Both the MSRE+HC workflow and the MSRE+mPCR workflow had excellent performance. In this particular experiment, the MSRE+HC workflow provided better performance than the MSRE+mPCR workflow (AUC 0.999 vs. 0.94). As shown in Table 2 and Figures 25A and 25B, higher sensitivity and specificity (sensitivity: 95%, specificity: 100%) were observed using HC compared to mPCR (sensitivity: 94%, specificity: 86%). In other experiments, the MSRE+mPCR workflow may provide better performance than the MSRE+HC workflow. [Table 2]

[0161] MSRE Target Assay Review To understand the characteristics of targeted assays that can provide a clearer distinction between CRC and healthy controls, we investigated the correlation between the average CRC / healthy control DOR ratio and the number of cleavage sites. Consistent with the findings in Example 2, assays with more MSRE cleavage sites provided greater discrimination (higher CRC / healthy control DOR ratios, Figure 26). We observed that not all assays with multiple MSRE cleavage sites provided a discriminatory high CRC / healthy control DOR ratio. It is possible that not all CpG sites within these target regions are hypermethylated in CRC.

[0162] Comparison of the top 40 targets between the MSRE+HC and MSRE+mPCR workflows Cross-checking the top 40 targets using HC with the top 40 targets from previous mPCR studies (Examples 1 and 2) using the same set of targets revealed that 24 / 40 HC targets were found in both mPCR studies and 36 / 40 were found in at least one mPCR study (Figure 27A), suggesting that assays with higher CRC / normal mDOR ratios are consistent between HC and mPCR workflows. Of the top 24 common good target assays, 23 / 24 targets contain 10 or more cleavage sites. One good assay contains 8 cleavage sites (Figure 27B). The majority of good assays utilize BstUI / HhaI / HpaII, consistent with the prevalence of MSRE assays.

[0163] In conclusion, the MSRE+HC approach can be used to effectively distinguish CRC-positive samples from CRC-negative samples or healthy control samples. The selection of methylation targets can be further improved by using information on the MSRE cleavage site and / or methylation load.

[0164] All references throughout this application, e.g., patent documents, published patent applications, including issued or granted patents or equivalents, and non-patent literature or other source materials, are incorporated by reference in their entirety herein, as if each reference were individually incorporated by reference, to the extent that each reference is at least partially consistent with the disclosure of this application (e.g., a partially conflicting reference is incorporated by reference except for the partially conflicting portion of the reference).

[0165] The terms and expressions employed herein are used as terms of description rather than limitation, and the use of such terms and expressions is not intended to exclude any equivalents of the shown and described features or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, while the present invention has been specifically disclosed by exemplary embodiments, representative embodiments, and optional features, it should be understood that optional features, modifications, and variations of the concepts disclosed herein may be utilized by those skilled in the art, and such modifications and variations are deemed to be within the scope of the invention as defined by the appended claims. The specific embodiments provided herein are representative of useful embodiments of the invention, and it will be apparent to those skilled in the art that the invention may be implemented using numerous variations of the devices, device components, and method steps described in this description. As will be apparent to those skilled in the art, the methods and devices useful for the present methods may include numerous optional compositions and processing elements and steps.

[0166] All patents and publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. The references cited herein are incorporated by reference in their entirety to indicate the state of the art as of their publication or filing date, and it is intended that this information may be incorporated herein, if necessary, to exclude specific aspects of the prior art. For example, if a composition of matter is claimed, it should be understood that compounds known and available in the art prior to the applicant's invention, including compounds for which the references cited herein provide useful disclosures, are not intended to be included in the composition of matter claims described herein.

[0167] Those skilled in the art will understand that starting materials, biological materials, reagents, synthetic methods, purification methods, analytical methods, assay methods, and biological methods other than those specifically exemplified can be employed in the practice of the present invention without resort to undue experimentation. All functional equivalents known to those of skill in the art of any such materials and methods are intended to be encompassed by the present invention. The terms and expressions employed are used as terms of description rather than limitation, and there is no intention in the use of such terms and expressions to exclude any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, while the present invention has been specifically disclosed by exemplary embodiments and optional features, it should be understood that optional features, modifications, and variations of the concepts disclosed herein may be utilized by those skilled in the art, and that such modifications and variations are deemed to be within the scope of the invention, as defined by the appended claims.

[0168] The disclosed embodiments, examples, and experiments are not intended to limit the scope of the present disclosure or to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. It should be understood that variations in the methods as described may be made without changing the fundamental aspects that the experiments are intended to illustrate.

[0169] Those skilled in the art may devise numerous modifications and other embodiments within the scope and spirit of the present disclosure. Indeed, variations in the described materials, methods, diagrams, experiments, examples, and embodiments may be made by those skilled in the art without changing the fundamental aspects of the present disclosure. Any of the disclosed embodiments may be used in combination with other disclosed embodiments.

[0170] In some cases, certain concepts have been described with reference to specific embodiments. However, those skilled in the art will recognize that various modifications and changes can be made without departing from the scope of the invention as set forth in the following claims. Accordingly, the specification and figures should be regarded in an illustrative rather than a restrictive sense, and all such modifications are intended to be included within the scope of the invention.

Claims

1. 1. A method for preparing a deoxyribonucleic acid (DNA) molecule useful for determining the methylation status of a genomic region of interest, said method comprising: ligating adaptors to sample DNA molecules obtained or derived from a first sample from a first subject, thereby forming a plurality of adapted DNA molecules, including adapted DNA molecules having methylation-sensitive restriction enzyme (MSRE) recognition sites; contacting the plurality of adapted DNA molecules with one or more MSREs, thereby generating MSRE-exposed adapted DNA molecules, wherein adapted DNA molecules exposed to at least a first plurality of MSREs having one or more unmethylated MSRE recognition sites are cleaved by at least one of said MSREs, and adapted DNA molecules exposed to a second plurality of MSREs are not cleaved by any of said MSREs, thus forming MSRE-exposed uncleaved adapted DNA molecules; and performing one or more amplification reactions to amplify one or more target regions from an uncleaved adapted DNA molecule or a copy thereof that has been exposed to an MSRE, wherein if the one or more target regions are present in the uncleaved adapted DNA molecule that has been exposed to the MSRE, a target region amplicon is formed.

2. The method of claim 1 , wherein each target region has one or more MSRE recognition sites.

3. 3. The method of claim 2, wherein the sample DNA molecules are circulating free DNA (cfDNA).

4. 4. The method of claim 3, wherein the one or more target region amplicons are generated by one or more polymerase chain reactions (PCR) using one or more primer pairs, wherein at least one primer of each of the one or more primer pairs is a target-specific primer.

5. 5. The method of claim 4, wherein the method further comprises detecting the one or more target region amplicons.

6. 6. The method of claim 5, wherein the one or more target regions is a set of target regions and the one or more primer pairs is a set of primer pairs, each configured to amplify one target region of the set.

7. 7. The method of claim 6, wherein the method further comprises quantifying the amount of at least one of the one or more target region amplicons.

8. 8. The method of claim 7, wherein the one or more target regions is a set of target regions and the one or more primer pairs is a set of primer pairs, each configured to amplify one target region of the set.

9. 4. The method of claim 3, wherein the one or more target region amplicons are generated by one or more ligation target capture (LTC) reactions using one or more probe-dependent primer pairs.

10. 10. The method of claim 9, wherein the method further comprises detecting the one or more target region amplicons.

11. 11. The method of claim 10, wherein the one or more target regions is a set of target regions and the one or more probe-dependent primer pairs is a set of probe-dependent primer pairs, each configured to amplify one target region of the set.

12. 11. The method of claim 10, wherein the method further comprises quantifying the amount of at least one of the one or more target region amplicons.

13. 13. The method of claim 12, wherein the one or more target regions is a set of target regions and the one or more primer pairs is a set of primer pairs, each configured to amplify one target region of the set.

14. 4. The method of claim 3, wherein performing the one or more amplifications comprises performing a universal amplification, wherein at least a plurality of the uncleaved adapted cfDNAs exposed to MSREs are amplified to generate amplicons, and the method further comprises selectively enriching one or more target regions from the amplicons using one or more hybrid capture probes to generate enriched target regions.

15. 15. The method of claim 14, wherein the method further comprises detecting the one or more enriched target regions.

16. 16. The method of claim 15, wherein selectively enriching the one or more target regions comprises selectively enriching a set of target regions, and wherein the one or more hybrid capture probes are a set of hybrid capture probes, each configured to selectively enrich one target region of the set.

17. 15. The method of claim 14, wherein the method further comprises quantifying the amount for at least one of the one or more enriched target regions.

18. 18. The method of claim 17, wherein the one or more target regions are a set of target regions and the one or more hybrid capture probes are a set of hybrid capture probes, each configured to selectively enrich for one target region of the set.

19. 1. A method for preparing a DNA molecule useful for determining the methylation status of a selected region of said DNA molecule, comprising: ligating adapters to circulating free DNA (cfDNA) obtained or derived from the first liquid sample, thereby forming a plurality of adapted cfDNAs, including adapted cfDNAs having MSRE recognition sites; contacting the plurality of adapted cfDNAs with one or more MSREs, thereby generating MSRE-exposed adapted cfDNAs, wherein the plurality of MSRE-exposed adapted cfDNAs are not cleaved by the one or more MSREs, thereby forming MSRE-exposed uncleaved adapted cfDNAs; performing a first amplification step in which at least a plurality of the uncleaved adapted cfDNAs exposed to the MSRE are amplified; performing a second amplification step to amplify a set of target regions from the amplified, MSRE-exposed, uncleaved, adapted cfDNA, each target region having one or more MSRE recognition sites, wherein a target region of the set is amplified if the target region is present in one or more of the amplified, MSRE-exposed, uncleaved, adapted cfDNA, thereby generating a target region amplicon; and quantifying the amount of at least some of the target region amplicons of the set by performing a next generation sequencing reaction on the clonally amplified target region amplicons or amplicons derived therefrom.

20. 20. The method of claim 19, wherein the first amplification step comprises one or more rounds of PCR using universal primers.

21. 21. The method of claim 20, wherein the second amplification step comprises one or more rounds of PCR using a set of primer pairs, each pair of the set designed to amplify a target region having one or more MSRE recognition sites, and at least one primer of each primer pair is a target-specific primer.

22. 21. The method of claim 20, wherein the second amplification step comprises one or more LTC reactions using a set of probe-dependent primer pairs, each pair of the set designed to amplify a target region having one or more MSRE recognition sites.

23. 1. A method for preparing a DNA molecule useful for determining the methylation status of a selected region of said DNA molecule, comprising: ligating adapters to circulating free DNA (cfDNA) obtained or derived from the first liquid sample, thereby forming a plurality of adapted cfDNAs, including adapted cfDNAs having MSRE recognition sites; contacting the plurality of adapted cfDNAs with one or more MSREs, thereby generating MSRE-exposed adapted cfDNAs, wherein the plurality of MSRE-exposed adapted cfDNAs are not cleaved by the one or more MSREs, thereby forming MSRE-exposed uncleaved adapted cfDNAs; amplifying at least a plurality of the MSRE-exposed uncleaved adaptor-attached cfDNAs; selectively enriching a subset of the amplified, MSRE-exposed, uncleaved, adapted cfDNA having one or more target regions using a set of hybrid capture probes, each target region containing one or more MSRE recognition sites, and each probe of the set is designed to hybridize to one target region; and quantifying the amount of at least some of the enriched subsets by performing a next generation sequencing reaction on the clonally amplified enriched subsets, or amplicons derived therefrom.

24. 24. The method of claim 23, wherein the amplifying comprises one or more rounds of PCR using universal primers.

25. 25. The method of claim 24, wherein the one or more target regions is a set of target regions.

26. 26. The method of any one of claims 1 to 25, wherein each target region comprises two or more recognition sites recognized by the one or more MSREs.

27. 25. The method of any one of claims 1 to 24, wherein each target region comprises three or more recognition sites recognized by the one or more MSREs.

28. 26. The method of any one of claims 1 to 25, wherein each target region comprises 1 to 10 recognition sites recognized by said one or more MSREs.

29. 26. The method of any one of claims 1 to 25, wherein each target region comprises 2 to 8 recognition sites recognized by said one or more MSREs.

30. 26. The method of any one of claims 1 to 25, wherein the one or more MSREs are 2 to 10 MSREs, and each target region comprises 2 to 10 recognition sites recognized by at least one of the MSREs.

31. The method of claim 1 , wherein the sample is a liquid sample.

32. 32. The method of claim 31 , wherein the liquid sample is a blood, plasma, serum, or urine sample.

33. 32. The method of claim 31 , wherein the liquid sample is a plasma sample.

34. 24. The method of claim 19 or claim 23, wherein quantifying the amount of at least some of the target region amplicons or at least some of the enriched subsets provides a quantitative value for the amount of circulating tumor DNA (ctDNA) in the first liquid sample.

35. 24. The method of claim 19 or claim 23, wherein the method further comprises performing an additional amplification reaction to amplify at least a portion of a target region from the target region amplicon or from an enriched subset of amplified, MSRE-exposed, uncleaved, adapted cfDNA having one or more target regions, wherein the additional amplification reaction is a clonal amplification reaction to form a clonally amplified target region amplicon, and wherein the NGS is performed on the clonally amplified target region amplicon.

36. 26. The method of any one of claims 1 to 25, wherein the adapted DNA molecule having one or more MSRE recognition sites or the adapted cfDNA having one or more MSRE recognition sites each comprises two or more MSRE recognition sites.

37. 14. The method of any one of claims 1 to 13, further comprising, prior to performing the one or more amplifications, performing universal PCR of the plurality of MSRE-exposed adapted DNA molecules or the plurality of MSRE-exposed adapted cfDNA using a universal PCR primer pair comprising a primer designed to bind to a universal primer binding sequence on an adapter to generate amplified MSRE-exposed adapted DNA molecules or MSRE-exposed adapted cfDNA.

38. 38. The method of claim 37, wherein at least one MSRE recognition site of the adapted DNA molecule or the adapted cfDNA having an MSRE recognition site is located outside the target region thereon.

39. 19. The method of any one of claims 14 to 18, wherein at least one of the MSRE-exposed adapted DNA molecule or the MSRE-exposed adapted cfDNA comprises one or more MSRE recognition sites outside of the target region of the MSRE-exposed adapted DNA molecule or the MSRE-exposed adapted cfDNA.

40. 23. The method of any one of claims 4 to 13, or 21 to 22, wherein both primers of at least one primer pair of the one or more primer pairs, the set of primer pairs, or the set of probe-dependent primer pairs are target-specific primers.

41. 23. The method of any one of claims 4 to 13, or 21 to 22, wherein both primers in a plurality of the one or more primer pairs, the set of primer pairs, or the set of probe-dependent primer pairs are target-specific primers.

42. 23. The method of any one of claims 4 to 13, or 21 to 22, wherein each primer of the one or more primer pairs, the set of primer pairs, or the set of probe-dependent primer pairs is a target-specific primer.

43. 23. The method of any one of claims 4 to 13, or 21 to 22, wherein at least one primer of at least one primer pair of the one or more primer pairs, the set of primer pairs, or the set of probe-dependent primer pairs is designed to bind to a primer binding site on one of the adapters.

44. 23. The method of any one of claims 4 to 13, or 21 to 22, wherein at least one primer of a plurality of primer pairs of the one or more primer pairs, the set of primer pairs, or the set of probe-dependent primer pairs is designed to bind to a primer binding site on one of the adapters.

45. 23. The method of any one of claims 4 to 13, or 21 to 22, wherein at least one primer of each primer pair of the one or more primer pairs, the set of primer pairs, or the set of probe-dependent primer pairs is designed to bind to a primer binding site on one of the adapters.

46. 26. The method of any one of claims 1 to 25, wherein one or more of the target regions, or set of target regions, comprises one or more MSRE recognition sites that overlap one or more of the primer binding sites, or set of primer binding sites.

47. 26. The method of any one of claims 1 to 25, wherein the primer pair is designed to bind to an uncleaved version of the adapted DNA molecule or the adapted cfDNA, but not to a cleaved adapted DNA molecule.

48. 26. The method of any one of claims 1 to 25, wherein the one or more MSREs are 2 to 5 MSREs, and each target region comprises 2 to 10 recognition sites recognized by at least one of the MSREs.

49. 26. The method of any one of claims 1 to 25, wherein the one or more MSREs are 2 to 5 MSREs, and each target region comprises 2 to 5 recognition sites recognized by at least one of the MSREs.

50. 3. The method of claim 1, wherein the first sample is a liquid sample.

51. 51. The method of claim 50, wherein the liquid sample is a blood, serum, plasma, urine, vitreous, sputum, saliva, tears, sweat, feces, bile, lymph, cervical mucus, or semen sample.

52. 51. The method of claim 50, wherein the liquid sample is a blood, plasma, serum, or urine sample.

53. 3. The method of claim 1 or 2, wherein the first sample is a blood sample or a derivative thereof.

54. 51. The method of claim 50, wherein the sample is a plasma sample.

55. 51. The method of claim 50, wherein the first sample comprises DNA from a tumor.

56. The method of claim 1 , wherein the first sample is a sample from cancer tissue.

57. 53. The method of any one of claims 1 to 52, wherein the first sample comprises DNA from a tumor.

58. 53. The method of any one of claims 1 to 52, wherein the first sample comprises DNA from a transplanted organ.

59. 53. The method of any one of claims 1 to 52, wherein the first sample comprises DNA from a fetus.

60. 55. The method of any one of claims 1 to 54, wherein the first subject is suspected of having or at risk of having a disease.

61. 61. The method of claim 60, wherein the disease is cancer.

62. The cancer is selected from the group consisting of ovarian cancer, soft tissue sarcoma, peripheral T-cell carcinoma, colorectal cancer, intrahepatic cholangiocarcinoma, glioblastoma, esophageal cancer, cutaneous T-cell lymphoma, non-Hodgkin's lymphoma, urothelial carcinoma, basal cell carcinoma, epithelioid sarcoma, pancreatic cancer, non-small cell lung cancer, Hodgkin's lymphoma, renal cell carcinoma, mesothelioma, metastatic uveal melanoma, kidney cancer, blood cancer, HER2-expressing cancer, non-melanoma skin cancer, liposarcoma, hepatocellular carcinoma, small lymphocytic lymphoma, Prostate cancer, breast cancer, anal cancer, marginal zone lymphoma, cutaneous squamous cell carcinoma, thyroid cancer, medullary thyroid cancer, triple-negative breast cancer, neuroendocrine prostate cancer, bladder cancer, paraganglioma, medulloblastoma, superficial basal cell carcinoma, head and neck squamous cell carcinoma, hematological malignancies, melanoma, B-cell lymphoma, relapsed / refractory acute myeloid leukemia, angiosarcoma, osteosarcoma, refractory cervical cancer, bile duct cancer, osteosarcoma, biliary tract cancer, castration-resistant prostate Cancer, gastroesophageal adenocarcinoma, rhabdomyosarcoma, carcinoma, non-muscle invasive bladder cancer, uveal melanoma, small cell lung cancer, cervical cancer, primary open angle glaucoma, follicular lymphoma, synovial sarcoma, liver cancer, carcinosarcoma, meningeal brain tumor, T cell lymphoma, lymphoma, small cell lung cancer, mantle cell lymphoma, B cell malignancies, endometrial cancer, myxoid / round cell liposarcoma, metastatic Merkel cell carcinoma, neuroblastoma, chronic lymphocytic leukemia 62. The method of claim 61, wherein the cancer is selected from hematologic malignancies, giant cell tumor of tendon sheath, sarcoma, acute myeloid leukemia, skin cancer, nasopharyngeal carcinoma, relapsed / refractory Ewing's sarcoma, bone cancer, glioma, salivary gland cancer, gastric cancer, benign tumors, low-grade serous ovarian cancer, metastatic breast cancer, multiple myeloma, diffuse large B-cell lymphoma, relapsed / refractory lymphoma, metastatic colorectal cancer, advanced malignancies, and acute lymphoblastic leukemia.

63. 62. The method of claim 61, wherein the cancer is selected from cancer of the abdomen or abdominal wall, adrenal gland, anus, appendix, bladder, bone, brain, breast, cervix, chest wall, colon, diaphragm, duodenum, ear, endometrium, esophagus, fallopian tube, gallbladder, gastroesophageal junction, head and neck, kidney, larynx, liver, lung, lymph node, malignant effusion, mediastinum, nasal cavity, omentum, ovary, pancreas, pancreaticobiliary region, parotid gland, pelvis, penis, pericardium, peritoneum, pleura, prostate, rectum, salivary gland, skin, small intestine, soft tissue, spleen, stomach, thyroid, tongue, trachea, ureter, uterus, vagina, vulva, or Whipple resection site.

64. 62. The method of claim 61, wherein the cancer is selected from lung cancer, breast cancer, bladder cancer, and colorectal cancer.

65. 62. The method of claim 61, wherein the methylation status of the set of target regions indicates the presence or absence of cancer.

66. 60. The method of any one of claims 25 to 59, wherein the subject is a pregnant woman.

67. 59. The method of any one of claims 25 to 58, wherein the subject is a subject comprising an organ from another individual.

68. 10. The method of any one of the preceding claims, wherein the method further comprises enriching the sample DNA molecules for DNA molecules that are between 70 and 500 base pairs in length.

69. 10. The method of any one of the preceding claims, wherein the method further comprises enriching the sample DNA molecules for DNA molecules that are between 100 and 200 base pairs in length.

70. 2. The method of any one of the preceding claims, wherein the method further comprises enriching the sample DNA molecules for DNA molecules that are between 130 and 170 base pairs in length.

71. 3. The method of claim 1, wherein the sample DNA molecules are fragmented to form fragmented DNA molecules before the ligation.

72. 10. The method of any one of the preceding claims, wherein the sample DNA molecules or the fragmented DNA molecules are exposed to one or more polymerases and / or kinases to produce the nucleic acid derivatives produced therefrom.

73. 10. The method of claim 1, wherein said ligating comprises ligating an adaptor to the nucleic acid derivative produced therefrom.

74. 10. The method of any one of the preceding claims, wherein the adapter is a Y adapter.

75. 10. The method of claim 1, wherein the adapters each comprise a universal priming site.

76. 10. The method of claim 1, wherein the adapter does not contain any MSRE recognition site.

77. 2. The method of any one of the preceding claims, wherein the one or more MSREs are selected from one or more of HpaII, HhaI, HpyCH41V, or BstU1.

78. 10. The method of any one of the preceding claims, wherein said contacting comprises contacting with two or more MSREs.

79. 79. The method of claim 78, wherein said contacting comprises contacting with two or more MSREs in a single reaction.

80. 10. The method of any one of the preceding claims, wherein said contacting comprises contacting with three or more MSREs.

81. 2. The method of claim 1, wherein said contacting comprises contacting with four or more MSREs.

82. 23. The method of any one of claims 4 to 13, or 21 to 22, wherein the set of primer pairs is a set of 2 to 1,000 primer pairs.

83. 23. The method of any one of claims 4 to 13, or 21 to 22, wherein at least one of the primer pairs comprises a universal primer and a target-specific primer.

84. 23. The method of any one of claims 4 to 13, or 21 to 22, wherein at least one of the primer pairs comprises two target-specific primers.

85. 23. The method of any one of claims 4 to 13, or 21 to 22, wherein at least one of the primers comprises a sample index.

86. 26. The method of any one of claims 1 to 25, wherein performing PCR further comprises using a primer that contains a sequence that can be used for a downstream sequencing reaction.

87. 22. The method of any one of claims 4 to 21, wherein performing PCR further comprises using primers that include a sample index.

88. 62. The method of claim 61, wherein the target regions each comprise a set of two or more MSRE recognition sites that are methylated in one or more cancers.

89. 62. The method of claim 61, wherein each of the target regions comprises three or more MSRE recognition sites that are predominantly methylated in cancer.

90. 17. The method of any one of claims 5-6, 10-11, or 15-16, wherein the method further comprises an additional amplification reaction that amplifies at least some of the amplified target region amplicons.

91. 91. The method of claim 90, wherein said additional amplification reaction is a clonal amplification reaction to form a clonally amplified target region amplicon.

92. 92. The method of Claim 91, wherein said detecting or quantifying comprises performing a sequencing reaction on said clonally amplified target region amplicons.

93. 17. The method of any one of claims 5-6, 10-11, or 15-16, wherein the detecting or quantifying comprises performing a sequencing reaction.

94. 94. The method of any one of claims 92 or 93, wherein the sequencing reaction is a next generation sequencing (NGS) reaction.

95. 95. The method of any one of claims 19-23 or 94, wherein the quantifying comprises counting sequence reads generated from the NGS reaction.

96. 96. The method of claim 95, wherein said quantifying comprises determining a read depth per target region for at least a portion of said target region.

97. 97. The method of claim 96, wherein the read depth of each of the target regions is normalized to the read depth of a normalization sequence.

98. 98. The method of claim 97, wherein the normalization sequence is a fully methylated DNA sequence.

99. 99. The method of claim 98, wherein the fully methylated DNA sequence is derived from pUC19.

100. 99. The method of claim 98, wherein the fully methylated DNA sequence is a synthetic sequence.

101. 98. The method of claim 97, wherein the normalizing sequence is a non-MSRE DNA sequence that does not have an MSRE recognition site for any of the one or more MSREs.

102. 98. The method of claim 97, wherein the non-MSRE DNA sequence is a human genomic DNA sequence.

103. 98. The method of claim 97, wherein the non-MSRE DNA sequence is a synthetic sequence.

104. 91. The method of claim 90, wherein the additional amplification reaction is a real-time PCR reaction.

105. 91. The method of claim 90, wherein the additional amplification reaction is a digital PCR reaction.

106. The method further comprises performing the method on a second sample or a second liquid sample; 10. The method of claim 1, wherein determining the methylation state of the set of target regions in the first sample further comprises comparing the amount of target region amplicons derived from the first sample with the amount of target region amplicons derived from the second sample.

107. 107. The method of claim 106, wherein the second sample is from a second subject.

108. 108. The method of claim 107, wherein the first subject is suspected of having or at risk of having a disease and the second subject is not suspected of having or at risk of having a disease.

109. 107. The method of claim 106, wherein the second sample is derived from a cell line.

110. 107. The method of claim 106, wherein the method is performed simultaneously on the first and second samples.

111. 107. The method of claim 106, wherein the second sample is from a first subject.

112. 112. The method of claim 111, wherein the second sample is taken at a first time point and the first sample is taken at a second time point.

113. 113. The method of claim 112, wherein the first time point precedes the second time point.

114. 26. The method of any one of claims 5 to 25, wherein said determining comprises comparing the amount quantified for the target region amplicon derived from the first sample to a preset value or an amount quantified for a target region amplicon derived from a second sample using the same method.

115. 26. The method of any one of claims 7-8, 12-13, 17-18, 19-22, and 23-25, wherein the method further comprises, in addition to a first performance of the method, a second performance of the method performed simultaneously with or at a different time than the first performance, wherein the second performance does not include a contacting step, and wherein the method comprises comparing the amount quantified in the first performance with the amount quantified in the second performance to determine the methylation state of the target nucleic acid.

116. 116. The method of claim 115, wherein said amount is no greater than the amount of amplicon produced using a negative control nucleic acid sample that does not have a methylated MSRE recognition site.

117. 116. The method of claim 115, wherein the amount is greater than the amount of amplicon produced using a negative control nucleic acid sample that does not have a methylated MSRE recognition site.

118. 10. The method of claim 1, wherein the method is capable of detecting fully methylated DNA molecules present at 1.0% or less (by mass) in a mixture of DNA molecules.

119. 10. The method of any one of the preceding claims, wherein the method is capable of distinguishing between samples having 1.0% or less (by mass) fully methylated DNA molecules and samples having 0% of said fully methylated DNA molecules.

120. 10. The method of any one of the preceding claims, wherein the method is capable of distinguishing between samples having 0.1% or less (by mass) fully methylated DNA molecules and samples having 0% of said fully methylated DNA molecules.

121. 10. The method of any one of the preceding claims, wherein the method is capable of distinguishing between samples having 0.05% or less (by mass) fully methylated DNA molecules and samples having 0% of said fully methylated DNA molecules.

122. 10. The method of any one of the preceding claims, wherein circulating tumor DNA (ctDNA) is present at a rate of 1% or less relative to the total circulating free DNA (cfDNA) in the sample.

123. 10. The method of any one of the preceding claims, wherein circulating tumor DNA (ctDNA) is present in a proportion of 0.1% or less of the total circulating free DNA (cfDNA) in the sample.

124. 10. The method of any one of the preceding claims, wherein circulating tumor DNA (ctDNA) is present in a proportion of 0.01% or less of the total circulating free DNA (cfDNA) in the sample.

125. 10. The method of any one of the preceding claims, wherein each of the adapters further comprises a molecular barcode.

126. 126. The method of Claim 125, wherein the number of adapters with different molecular barcodes is between 10 and 1,000, and wherein the ratio of the total number of sample DNA or cfDNA molecules to the number of different molecular barcodes in the ligation reaction is at least 1,000:

1.

127. 126. The method of Claim 125, wherein the ratio of the total number of sample DNA or cfDNA molecules to the number of different molecular barcodes in the ligation reaction is at least 10,000:

1.

128. 10. The method of claim 1, wherein the sample DNA obtained or derived from the first sample comprises a mixture of hypermethylated and hypomethylated DNA.

129. 129. The method of claim 128, wherein the adaptors are ligated to a mixture of hypermethylated and hypomethylated DNA.

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