Methods for enriching for methylated nucleic acid molecules

Abstract

Disclosed are methods for enriching for nucleic acid molecules (e.g., enriching for methylated nucleic acid molecules), thereby enriching for target sequences of interest in a sample (e.g., a sample obtained from a subject). Generally, methods involve use of nucleic acid blockers with termination modifications that block or reduce the amplification of candidate sequences e.g., sequences of non-methylated nucleic acid molecules. Such methods are useful for enriching for a signal in a sample, such as a signal informative for determining presence or absence of cancer in the sample.

Description

Attorney Docket No. HRG-027WOMETHODS FOR ENRICHING FOR METHYLATED NUCLEIC ACID MOLECULESCROSS REFERENCE

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 708358, filed October 17, 2024, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.BACKGROUND

[0002] Non-invasive liquid biopsy tests are proving to be the next frontier in cancer diagnostics. There is a rising interest in sensitive, low-cost liquid biopsy assays to detect cancer-specific methylation events. Real-time quantitative methylation-specific PCR (qMSP) allows for the detection of rare methylated fragments; however widespread application has not been achieved. While qMSP achieves high specificity and sensitivity with low cfDNA inputs, assay design is challenged by the mostly three- base genome of bisulfite-converted DNA and the high homology between methylated ctDNA and excessive unmethylated cfDNA background.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on October 7, 2025, is named HRG-027WO_SL.xml and is 12,635 bytes in size.SUMMARY OF THE INVENTION

[0004] The disclosure relates to methods of enriching target sequences in a nucleic acid sample. The target sequence may be indicative of the risk of developing or the presence of cancer in the subject from whom the sample was taken. Disclosed herein are methods that improve detection of nucleic acids containing a target sequence, e.g., rare target sequences, by isolating and / or enriching such target sequences in a nucleic acid sample. For example, the rare target sequence may be in a nucleic acid sequence from a cfDNA sample, such as a cfDNA sample that has been treated with bisulfite or chemical / enzymatic conversion to convert cytosines to uracils to preserve information regarding the methylation status of a particular nucleic acid sequence (e.g.,Attorney Docket No. HRG-027WO comprising a CpG site), in a subject. Disclosed herein are blockers (e.g., blockers with a polymerase terminating modification) that bind to candidate sequences (e.g., sequences derived from non-methylated CpG sites) to inhibit the amplification and / or detection of such sequences with high specificity, while maintaining qMSP sensitivity in low copies of target DNA (e.g., sequences derived from methylated CpG sites). The combination of qMSP and blockers with a polymerase terminating modification enables detection of low levels of cancer-specific methylation patterns and estimation of percent methylated DNA.

[0005] Disclosed herein is a method for performing differential enrichment of nucleic acid molecules, the method comprising: a) providing a mixture of nucleic acid molecules comprising: a first set of nucleic acid molecules comprising a target sequence derived from a sequence comprising one or more methylated CpG sites; and a second set of nucleic acid molecules comprising a candidate sequence derived from a sequence comprising one or more nonmethylated CpG sites, b) providing a blocker that binds to the candidate sequence, or a portion thereof, or the complementary sequence thereof, of the second set of nucleic acid molecules, the blocker comprising a polymerase terminating modification; c) selectively enrich for the first set of nucleic acid molecules comprising the target sequence in comparison to the second set of nucleic acid molecules comprising the candidate sequence, wherein the blocker comprising the polymerase terminating modification prevents or reduces enrichment of the second set of nucleic acid molecules comprising the candidate sequence; and d) detecting the selectively enriched first set of nucleic acid molecules.

[0006] In some embodiments, the polymerase terminating modification is selected from the group consisting of 3’ ddC, 3’ ddG, 3’ ddA, 3’ ddT, 3’ inverted dT, 3’ C3 spacer, 3’ amino, and 3’ phosphorylation. In some embodiments, the polymerase terminating modification is 3’ ddC. In some embodiments, the polymerase terminating modification is 3’ C3.

[0007] In some embodiments, the candidate sequence comprises at least two unmethylated CpG sites or the complementary sequence thereof. In some embodiments, the candidate sequence comprises two unmethylated CpG sites or the complementary sequence thereof. In some embodiments, the candidate sequence comprises three unmethylated CpG sites or the complementary sequence thereof.

[0008] In some embodiments, selectively enriching the first set of nucleic acid molecules comprises performing one or more of PCR, RT-PCR, digital PCR, qPCR, nicking endonucleaseAttorney Docket No. HRG-027WO amplification (NEAR), transcription-mediated amplification (TMA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HAD), strand displacement amplification (SDA), sequencing library PCR, hybrid capture, microarrays, next generation sequencing (NGS), or hybrid capture PCR.

[0009] In some embodiments, selectively enriching for the first set of nucleic acid molecules comprises providing a forward primer and a reverse primer for performing nucleic acid amplification.

[0010] In some embodiments, when the forward primer or the reverse primer is bound to a sequence of the second set of nucleic acid molecules, an end of the forward primer or the reverse primer overlaps with the sequence bound by the blocker by at least 3 nucleotides, at least 5 nucleotides, at least 7 nucleotides, at least 9 nucleotides, or at least 11 nucleotides.

[0011] In some embodiments, the forward primer or the reverse primer is bound to a sequence of the second set of nucleic acid molecules, an end of the forward primer or the reverse primer overlaps with the sequence bound by the blocker by 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, or 11 nucleotides.

[0012] In some embodiments, when the forward primer or the reverse primer is bound to a sequence of the second set of nucleic acid molecules, an end of the forward primer or the reverse primer overlaps with the sequence bound by the blocker by 7 nucleotides.

[0013] In some embodiments, when the forward primer or the reverse primer is bound to a sequence of the second set of nucleic acid molecules, an end of the forward primer or the reverse primer overlaps with the sequence bound by the blocker by 10 nucleotides.

[0014] In some embodiments, the blocker comprises a sequence that shares at least 85% identity with any one of SEQ ID NOs: 1-6. In some embodiments, the blocker comprises a sequence that shares at least 95% identity with any one of SEQ ID NOs: 1-6.

[0015] In some embodiments, the blocker, when bound to the candidate sequence, has a melting temperature at least 5°C higher, at least 6°C higher, at least 7°C, or at least 8°C higher than the melting temperature of the forward primer or the reverse primer.

[0016] In some embodiments, the blocker, when bound to the candidate sequence, has a melting temperature at least 6°C higher than the melting temperature of the forward primer or the reverse primer. In some embodiments, the blocker, when bound to the candidate sequence, has a melting temperature at least 7°C higher than the melting temperature of the forward primer or the reverseAttorney Docket No. HRG-027WO primer. In some embodiments, the blocker, when bound to the candidate sequence, has a melting temperature at least 8°C higher than the melting temperature of the forward primer or the reverse primer.

[0017] In some embodiments, the blocker comprises between 20 and 35 nucleosides, optionally between 23-33 nucleosides.

[0018] In some embodiments, the method further comprises, between step (b) and step (c), exposing the blocker and the candidate sequence to one or more temperatures, thereby enabling the blocker to bind to the candidate sequence or complementary sequence thereof.

[0019] In some embodiments, wherein the one or more temperatures comprises a temperature between 50°C and 65°C, optionally about 56°C.

[0020] In some embodiments, the first set of nucleic acid molecules and / or the second set of nucleic acid molecules are DNA. In some embodiments, the DNA is cell-free DNA.

[0021] In some embodiments, the first set of nucleic acid molecules and / or the second set of nucleic acid molecules are RNA.

[0022] In some embodiments, the first set of nucleic acid molecules and / or the second set of nucleic acid molecules have been treated using bisulfite conversion to convert unmethylated cytosines to uracil. In some embodiments, the first set of nucleic acid molecules and / or the second set of nucleic acid molecules have been treated using enzymatic conversion to convert unmethylated cytosines to uracil. In some embodiments, the enzymatic conversion is selected from TET2 oxidation of cytosines and APOBEC conversion of cytosines.

[0023] In some embodiments, the first set of nucleic acid molecules and / or the second set of nucleic acid molecules were obtained from a sample. In some embodiments, the sample comprises a tissue sample, a blood sample, a stool sample, a urine sample, a mucous sample, or a saliva sample.

[0024] In some embodiments, the target sequence comprises at least a CpG island, or a portion thereof.

[0025] In some embodiments, the method further comprises either simultaneous with step (b) or after step (b), providing a second blocker that binds to a different candidate sequence, or a portion thereof, of the second set of nucleic acid molecules, the second blocker comprising a polymerase terminating modification.Attorney Docket No. HRG-027WO

[0026] In some embodiments, the method further comprises: either simultaneous with step (b) or after step (b), providing a probe capable of hybridizing to one or both of the target sequence, or a portion thereof, of the first set of nucleic acid molecules and the candidate sequence, or a portion thereof, of the second set of nucleic acid molecules.

[0027] In some embodiments, the method further comprises hybridizing the probe to the target sequence, or a portion thereof of the first set of nucleic acid molecules, wherein selectively enriching for the first set of nucleic acid molecules comprises enriching for the first set of nucleic acid molecules using the probe hybridized to the target sequence, or a portion thereof, of the first set of nucleic acid molecules.

[0028] In some embodiments, enriching for the first set of nucleic acid molecules using the probe comprises performing hybrid capture.

[0029] Disclosed herein is a method for performing differential enrichment of nucleic acid molecules, the method comprising: a) providing nucleic acid molecules comprising a target sequence derived from a sequence comprising one or more methylated CpG sites; b) providing a blocker capable of binding to a candidate sequence, or a portion thereof, or the complementary sequence thereof, the candidate sequence only differing from the target sequence at nucleotides corresponding to the one or more methylated CpG sites, wherein the blocker comprises a polymerase terminating modification; c) enriching for the nucleic acid molecules comprising the target sequence, wherein the blocker comprising a polymerase terminating modification does not prevent enrichment of the nucleic acid molecules comprising the target sequence; and d) detecting the enriched nucleic acid molecules.

[0030] Disclosed herein is a method for performing differential enrichment of nucleic acid molecules, the method comprising: a) providing nucleic acid molecules comprising a candidate sequence derived from a sequence comprising one or more non-methylated CpG sites; b) providing a blocker capable of binding to the candidate sequence, or a portion thereof, or the complementary sequence thereof, wherein the blocker comprises a polymerase terminating modification; and c) exposing the nucleic acid molecules comprising the candidate sequence to conditions suitable for nucleic acid enrichment, wherein the blocker comprising a polymerase terminating modification binds to the candidate sequence, or a portion thereof, or the complementary sequence thereof, and prevents or reduces enrichment of nucleic acid molecules comprising the candidate sequence.Attorney Docket No. HRG-027WO

[0031] These and other aspects and features of the disclosure are described in the following detailed description and claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The foregoing and other objects, features and advantages of the invention will become apparent from the following description of preferred embodiments, as illustrated in the accompanying drawings. Like referenced elements identify common features in the corresponding drawings. The drawings are not necessarily to scale, with emphasis instead being placed on illustrating the principles of the present invention, in which:

[0033] It is noted that wherever practicable similar or like reference numbers may be used in the figures and may indicate similar or like functionality. For example, a letter after a reference numeral, such as “nucleic acid molecule 415 A,” indicates that the text refers specifically to the element having that particular reference numeral. A reference numeral in the text without a following letter, such as “nucleic acid molecule 415” refers to any or all of the elements in the figures bearing that reference numeral (e. g. “nucleic acid molecule 415” in the text refers to reference numerals “nucleic acid molecule 415A” and / or “nucleic acid molecule 415B” in the figures).

[0034] Figure (FIG) 1 shows an example flow diagram for differentially enriching for nucleic acid molecules, in accordance with an embodiment.

[0035] FIG. 2A depicts an example conversion of nucleic acids, in accordance with an embodiment.

[0036] FIG. 2B shows the results of nitrite conversion on select nucleotides, in accordance with a second embodiment. Figure adapted from Li et al. (2022) Genome Biology 23: 122.

[0037] FIG. 3A depicts diagrams involving blockers with terminating modifications, in accordance with an embodiment.

[0038] FIG. 3B depicts a diagram involving provision of probes, in accordance with a first embodiment.

[0039] FIG. 3C depicts a diagram involving provision of primers, in accordance with a second embodiment.Attorney Docket No. HRG-027WO

[0040] FIG. 3D depicts a diagram involving provision of primers and probes, in accordance with a third embodiment.

[0041] FIG. 4A shows an example depiction of blocker-induced enrichment.

[0042] FIG. 4B shows changes in cycle threshold (Ct) for methylated DNA does not change in the presence or absence of blockers. FIG 4C shows a shift in the Ct value for unmethylated DNA in the presence of blockers. FIG. 4D depicts a scenario in which unmethylated DNA is not amplified, thereby resulting in a significant change in Ct value.

[0043] FIG. 5 shows an exemplary gel comparing no blocker and presence of Blocker #8, #9, or #10, with a C3 spacer, in the amplification of unmethylated CpG4 (-) gBlock.

[0044] FIG. 6A shows an exemplary gel of qMSP reactions run across various Tm in the absence of a blocker.

[0045] FIG. 6B shows an exemplary gel of qMSP reactions run across various Tm in the present of Blocker #10 with a C3 spacer.

[0046] FIG. 7 shows exemplary gels of the comparison of qMSP reactions with Blocker #10 using original full length BSP primers and shortened BSP primers at a range of Tm.

[0047] FIG. 8 shows exemplary gels comparing qMSP reactions with Blocker #10 using unmethylated, converted gBlock DNA at the indicated ratios of blocker to primer across a range of Tm.

[0048] FIG. 9 shows exemplary gels of using Blocker #10 with an MSP probe or BSP probe at various annealing temperatures.DETAILED DESCRIPTIONDefinitions

[0049] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0050] As used herein, the term “about” or “approximately” can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which can depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per theAttorney Docket No. HRG-027WO practice in the art. “About” can mean a range of ±20%, ±10%, ±5%, or ±1% of a given value. The term “about” or “approximately” can mean within an order of magnitude, within 5-fold, or within 2-fold, of a value. Where a particular value is described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value can be assumed. The term “about” can have the meaning as commonly understood by one of ordinary skill in the art. The term “about” can refer to ±10%. The term “about” can refer to ±5%.

[0051] It should be understood that the expression of “at least one of’ includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression “and / or” in connection with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context.

[0052] As used herein, the term “biological sample,” or “sample” refers to any sample taken from a subject, which can reflect a biological state associated with the subject, and that includes cell free DNA. A biological sample can take any of a variety of forms, such as a liquid biopsy (e.g., blood, urine, stool, saliva, or mucous), or a tissue biopsy, or other solid biopsy. Examples of biological samples include, but are not limited to, blood, whole blood, plasma, serum, urine, cerebrospinal fluid, fecal, saliva, sweat, tears, pleural fluid, pericardial fluid, or peritoneal fluid of the subject. A biological sample can include any tissue or material derived from a living or dead subject. A biological sample can be a cell-free sample. A biological sample can comprise a nucleic acid (e.g., DNA or RNA) or a fragment thereof. The term “nucleic acid” can refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or any hybrid or fragment thereof. The nucleic acid in the sample can be a cell-free nucleic acid. A sample can be a liquid sample or a solid sample (e.g., a cell or tissue sample). A biological sample can be a bodily fluid, such as blood, plasma, serum, urine, vaginal fluid, fluid from a hydrocele (e.g., of the testis), vaginal flushing fluids, pleural fluid, ascitic fluid, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchoalveolar lavage fluid, discharge fluid from the nipple, aspiration fluid from different parts of the body (e.g., thyroid, breast), etc. A biological sample can be a stool sample. In various embodiments, the majority of DNA in a biological sample that has been enriched for cell-free DNA (e.g., a plasma sample obtained via a centrifugation protocol) can be cell-free (e.g., greater than 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the DNA can be cell-free). A biologicalAttorney Docket No. HRG-027WO sample can be treated to physically disrupt tissue or cell structure (e.g., centrifugation and / or cell lysis), thus releasing intracellular components into a solution which can further contain enzymes, buffers, salts, detergents, and the like which can be used to prepare the sample for analysis.

[0053] As used herein, the terms “nucleic acid” and “nucleic acid molecule” are used interchangeably. The terms refer to nucleic acids of any composition form, such as deoxyribonucleic acid (DNA, e.g., complementary DNA (cDNA), genomic DNA (gDNA) and the like), and / or DNA analogs (e.g., containing base analogs, sugar analogs and / or a non-native backbone and the like), all of which can be in single- or double-stranded form. Unless otherwise limited, a nucleic acid can comprise known analogs of natural nucleotides, some of which can function in a similar manner as naturally occurring nucleotides. A nucleic acid can be in any form useful for conducting processes herein (e.g., linear, circular, supercoiled, single-stranded, double-stranded and the like). A nucleic acid in some embodiments can be from a single chromosome or fragment thereof (e.g., a nucleic acid sample may be from one chromosome of a sample obtained from a diploid organism). In certain embodiments nucleic acids comprise nucleosomes, fragments or parts of nucleosomes or nucleosome-like structures. Nucleic acids can comprise protein (e.g., histones, DNA binding proteins, and the like). Nucleic acids analyzed by processes described herein can be substantially isolated and are not substantially associated with protein or other molecules. Nucleic acids can also include derivatives, variants and analogs of DNA synthesized, replicated or amplified from single-stranded (“sense” or “antisense,” “plus” strand or “minus” strand, “forward” reading frame or “reverse” reading frame) and doublestranded polynucleotides. Deoxyribonucleotides can include deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine. A nucleic acid may be prepared using a nucleic acid obtained from a subject as a template.

[0054] As used herein, the terms “template nucleic acid” and “template nucleic acid molecule(s)” are used interchangeably. The terms refer to nucleic acid that has been obtained from a sample and processed to form an immortalized library. The template nucleic acid can be nucleic acid obtained directly from the sample, or nucleic acid that is derived from that obtained directly from the sample. Examples of nucleic acid derived from a sample include DNA that has been reverse-transcribed from RNA obtained directly from a sample, or DNA that has be amplified from DNA obtained directly from a sample, for example, by PCR.Attorney Docket No. HRG-027WO

[0055] As used herein, the term “cell-free nucleic acids” refers to nucleic acid molecules that can be found outside cells, in bodily fluids such as blood, whole blood, plasma, serum, urine, cerebrospinal fluid, fecal, saliva, sweat, sweat, tears, pleural fluid, pericardial fluid, or peritoneal fluid of a subject. Cell-free nucleic acids originate from one or more healthy cells and / or from one or more cancer cells, or from non-human sources such bacteria, fungi, viruses. Examples of the cell-free nucleic acids include but are not limited to cell-free DNA (“cfDNA”), including mitochondrial DNA or genomic DNA, and cell-free RNA. In certain embodiments herein, instruments for assessing the quality of the cell-free nucleic acids, such as the TapeStation System from Agilent Technologies (Santa Clara, CA) can be used. Concentrating low-abundance cfDNA can be accomplished, for example using a Qubit™ Fluorometer from Thermofisher Scientific (Waltham, MA).

[0056] As used herein, the term “methylation” refers to a modification of a nucleic acid where a hydrogen atom on the pyrimidine ring of a cytosine base is converted to a methyl group, forming 5-methylcytosine. Methylation can occur at dinucleotides of cytosine and guanine referred to herein as “CpG sites.” Methylation of cytosine can occur in cytosines in other sequence contexts, for example, 5'-CHG-3' and 5'-CHH-3', where H is adenine, cytosine or thymine. Cytosine methylation can also be in the form of 5-hydroxymethylcytosine. Methylation of DNA can include methylation of non-cytosine nucleotides, such as N6-methyladenine. Anomalous cfDNA methylation can be identified as hypermethylation or hypomethylation, both of which may be indicative of cancer status. As is well known in the art, DNA methylation anomalies (compared to healthy controls) can cause different effects, which may contribute to cancer.

[0057] Certain portions of a genome comprise regions with a high frequency of CpG sites. A CpG site is portion of a genome that has cytosine and guanine separated by only one phosphate group and is often denoted as “5’ — C — phosphate — G — 3',” or “CpG” for short. Regions with a high frequency of CpG sites are commonly referred to as “CG islands” or “CGIs.” It has been found that certain CGIs and certain features of certain CGIs in tumor cells tend to be different from the same CGIs or features of the CGIs in healthy cells. Herein, such CGIs and features of the genome are referred to herein as “cancer informative CGIs,” which is defined and described in more detail below. An “informative CpG” can be specified by reference to a specific CpG site, or to a collection of one or more CpG sites by reference to a CG island that contains the collection. These cancer informative CGIs tend to have methylation patterns in tumor cells thatAttorney Docket No. HRG-027WO are different from the methylation patterns in healthy cells. DNA fragments from other CGIs may not express such differences.

[0058] As used herein, “DNA methylation” in mammalian genomes can refer to the addition of a methyl group to position 5 of the heterocyclic ring of cytosine (e.g., to produce 5- methylcytosine) among CpG dinucleotides. Methylation of cytosine can occur in cytosines in other sequence contexts, for example, 5’-CHG-3’ and 5’-CHH-3’, where H is adenine, cytosine or thymine. Cytosine methylation can also be in the form of 5 -hydroxymethylcytosine. Methylation of DNA can include methylation of non-cytosine nucleotides, such as N6- methyladenine.

[0059] The phrase “target sequence” refers to a sequence of a nucleic acid derived from a sequence comprising one or more methylated CpG sites. For example, the target sequence may be a sequence of a converted nucleic acid (e.g., where unmethylated cytosines have been converted to uracil and / or where methylated cytosines remain cytosines). In various embodiments, a target sequence includes one, two, three, four, five, six, seven, eight, nine, or ten CpG sites. In various embodiments, a target sequence includes one or more CpG sites within a region disclosed in Table 1 or Table 2. In particular embodiments, a target sequence includes five CpG sites within a region disclosed in Table 1 or Table 2. In particular embodiments, a target sequence includes five sequential CpG sites within a region disclosed in Table 1 or Table 2.

[0060] The phrase “candidate sequence” refers to a sequence of a nucleic acid derived from a sequence comprising one or more non-methylated CpG sites. For example, the candidate sequence may be a sequence of a converted nucleic acid (e.g., where unmethylated cytosines have been converted to uracil and / or where methylated cytosines remain cytosines). In various embodiments, a candidate sequence includes one, two, three, four, five, six, seven, eight, nine, or ten CpG sites. In various embodiments, a candidate sequence includes one or more CpG sites within a region disclosed in Table 1 or Table 2. In particular embodiments, a candidate sequence includes five CpG sites within a region disclosed in Table 1 or Table 2. In particular embodiments, a candidate sequence includes five sequential CpG sites within a region disclosed in Table 1 or Table 2.

[0061] The phrase “sequential CpG sites” refers to CpG sites within a range of genomic locations in which all CpG sites within the range of genomic locations are part of the sequentialAttorney Docket No. HRG-027WOCpG sites. Sequential CpG sites include a neighboring CpG site, i.e., a previous contiguous or next contiguous CpG site.

[0062] The phrase “unmethylated nucleic acid molecules” is broadly used to encompass nucleic acid molecules comprising sequences that include one or more unmethylated CpG sites and / or nucleic acid sequences derived from nucleic acid sequences that include one or more unmethylated CpG sites. For example, “unmethylated nucleic acid molecules” can refer to converted nucleic acid sequences (e.g., bisulfite-converted nucleic acid sequences) that are derived from cell-free DNA that include sequences with one or more unmethylated CpG sites.

[0063] As used herein, the term “amplifying” means performing an amplification reaction. In one aspect, an amplification reaction is “template-driven” in that base pairing of reactants, either nucleotides or oligonucleotides, have complements in a template polynucleotide that are required for the creation of reaction products. In one aspect, template-driven reactions are primer extensions with a nucleic acid polymerase, or oligonucleotide ligations with a nucleic acid ligase. Such reactions include, but are not limited to, polymerase chain reactions (PCRs), bisulfitespecific qPCR (qBSP), methylation-specific qPCR (qMSP), linear polymerase reactions, nucleic acid sequence-based amplification (NASBAs), rolling circle amplifications, and the like, disclosed in the following references, each of which are incorporated herein by reference herein in their entirety: Mullis et al. , U. S. Pat. Nos. 4,683,195; 4,965,188; 4,683,202; 4,800,159 (PCR); Gelfand et al. , U. S. Pat. No. 5,210,015 (real-time PCR with “taqman” probes); Wittwer et al. , U. S. Pat. No. 6,174,670; Kacian et al. , U. S. Pat. No. 5,399,491 (“NASBA”); Lizardi, U. S. Pat. No. 5,854,033; Aono et al. , Japanese patent publ. JP 4-262799 (rolling circle amplification); and the like. As used herein, bisulfite-specific PCR refers to PCR that amplifies converted DNA with no or limited methylation bias. As used herein, methylation-specific PCR refers to PCR that is methylation-specific and bi-sulfite specific, which amplifies converted methylated DNA. In one aspect, the amplification reaction is PCR. An amplification reaction may be a “real-time” amplification if a detection chemistry is available that permits a reaction product to be measured as the amplification reaction progresses, e.g., “real-time PCR,” or “realtime NASBA” as described in Leone et al., Nucleic Acids Research, 26: 2150-2155 (1998), and like references.

[0064] The terms “fragment” or “segment,” as used interchangeably herein, refer to a portion of a larger polynucleotide molecule. A polynucleotide, for example, can be broken up, orAttorney Docket No. HRG-027WO fragmented into, a plurality of segments. Various methods of fragmenting nucleic acid are well known in the art. These methods may be, for example, either chemical or physical or enzymatic in nature. Enzymatic fragmentation may include partial degradation with a DNase; partial depurination with acid; the use of restriction enzymes; intron-encoded endonucleases; DNA- based cleavage methods, such as triplex and hybrid formation methods, that rely on the specific hybridization of a nucleic acid segment to localize a cleavage agent to a specific location in the nucleic acid molecule; or other enzymes or compounds which cleave a polynucleotide at known or unknown locations. Physical fragmentation methods may involve subjecting a polynucleotide to a high shear rate. High shear rates may be produced, for example, by moving DNA through a chamber or channel with pits or spikes, or forcing a DNA sample through a restricted size flow passage, e.g., an aperture having a cross sectional dimension in the micron or submicron range. Other physical methods include sonication and nebulization. Combinations of physical and chemical fragmentation methods may likewise be employed, such as fragmentation by heat and ion-mediated hydrolysis. See, e.g., Sambrook et al., “Molecular Cloning: A Laboratory Manual,” 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (2001) (“Sambrook et al. ) which is incorporated herein by reference for all purposes. These methods can be optimized to digest a nucleic acid into fragments of a selected size range.

[0065] The terms “polymerase chain reaction” or “PCR,” as used interchangeably herein, mean a reaction for the in vitro amplification of specific DNA sequences by the simultaneous primer extension of complementary strands of DNA. In other words, PCR is a reaction for making multiple copies or replicates of a target nucleic acid flanked by primer binding sites, such reaction comprising one or more repetitions of the following steps: (i) denaturing the target nucleic acid, (ii) annealing primers to the primer binding sites, and (iii) extending the primers by a nucleic acid polymerase in the presence of nucleoside triphosphates. Usually, the reaction is cycled through different temperatures optimized for each step in a thermal cycler instrument. Particular temperatures, durations at each step, and rates of change between steps depend on many factors that are well-known to those of ordinary skill in the art, e.g., exemplified by the following references: McPherson et al., editors, PCR: A Practical Approach and PCR2: A Practical Approach (IRL Press, Oxford, 1991 and 1995, respectively). For example, in a conventional PCR using Taq DNA polymerase, a double stranded target nucleic acid may be denatured at a temperature>90° C, primers annealed at a temperature in the range 50-75°C, andAttorney Docket No. HRG-027WO primers extended at a temperature in the range 72-78°C. The term “PCR” encompasses derivative forms of the reaction, including, but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, and the like. The particular format of PCR being employed is discernible by one skilled in the art from the context of an application. Reaction volumes can range from a few hundred nanoliters, e.g., 200 nL, to a few hundred microliters, e.g., 200 pL. “Reverse transcription PCR,” or “RT-PCR,” means a PCR that is preceded by a reverse transcription reaction that converts a target RNA to a complementary single stranded DNA, which is then amplified, an example of which is described in Tecott et al. , U. S. Pat. No. 5,168,038, the disclosure of which is incorporated herein by reference in its entirety. “Real-time PCR” means a PCR for which the amount of reaction product, i. e., amplicon, is monitored as the reaction proceeds. There are many forms of real-time PCR that differ mainly in the detection chemistries used for monitoring the reaction product, e.g., Gelfand et al. , U. S. Pat. No. 5,210,015 (“taqman”); Wittwer et al. , U. S. Pat. Nos. 6,174,670 and 6,569,627 (intercalating dyes); Tyagi et al. , U. S. Pat. No. 5,925,517 (molecular beacons); the disclosures of which are hereby incorporated by reference herein in their entireties. Detection chemistries for real-time PCR are reviewed in Mackay et al., Nucleic Acids Research, 30: 1292-1305 (2002), which is also incorporated herein by reference. “Nested PCR” means a two-stage PCR wherein the amplicon of a first PCR becomes the sample for a second PCR using a new set of primers, at least one of which binds to an interior location of the first amplicon. As used herein, “initial primers” in reference to a nested amplification reaction mean the primers used to generate a first amplicon, and “secondary primers” mean the one or more primers used to generate a second, or nested, amplicon. “Asymmetric PCR” means a PCR wherein one of the two primers employed is in great excess concentration so that the reaction is primarily a linear amplification in which one of the two strands of a target nucleic acid is preferentially copied. The excess concentration of asymmetric PCR primers may be expressed as a concentration ratio. Typical ratios are in the range of from 10 to 100. “Multiplexed PCR” means a PCR wherein multiple target sequences (or a single target sequence and one or more reference sequences) are simultaneously carried out in the same reaction mixture, e.g., Bernard et al., Anal. Biochem, 273: 221-228 (1999) (two-color real-time PCR). Usually, distinct sets of primers are employed for each sequence being amplified. Typically, the number of target sequences in a multiplex PCR is in the range of from 2 to 50, or from 2 to 40, or from 2 to 30. In particular embodiments, the number of targetAttorney Docket No. HRG-027WO sequences in a multiplex PCR is about 4. “Quantitative PCR” means a PCR designed to measure the abundance of one or more specific target sequences in a sample or specimen. Quantitative PCR includes both absolute quantitation and relative quantitation of such target sequences. Quantitative measurements are made using one or more reference sequences or internal standards that may be assayed separately or together with a target sequence. The reference sequence may be endogenous or exogenous to a sample or specimen, and in the latter case, may comprise one or more competitor templates. Typical endogenous reference sequences include segments of transcripts of the following genes: 0-actin, GAPDH, P2-microglobulin, ribosomal RNA, and the like. Techniques for quantitative PCR are well-known to those of ordinary skill in the art, as exemplified in the following references, which are incorporated by reference herein in their entireties: Freeman et al. , Biotechniques, 26: 112-126 (1999); Becker-Andre et al. , Nucleic Acids Research, 17: 9437-9447 (1989); Zimmerman et al. , Biotechniques, 21 : 268-279 (1996); Diviacco et al. , Gene, 122: 3013-3020 (1992); and Becker- Andre et al. , Nucleic Acids Research, 17: 9437-9446 (1989).

[0066] The term “primer” as used herein means an oligonucleotide, either natural or synthetic, that is capable, upon forming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3' end along the template so that an extended duplex is formed. Extension of a primer is usually carried out with a nucleic acid polymerase, such as a DNA or RNA polymerase. The sequence of nucleotides added in the extension process is determined by the sequence of the template polynucleotide. Usually, primers are extended by a DNA polymerase. Primers usually have a length in the range of from 14 to 40 nucleotides, or in the range of from 18 to 36 nucleotides. Primers are employed in a variety of nucleic amplification reactions, for example, linear amplification reactions using a single primer, or polymerase chain reactions, employing two or more primers. Guidance for selecting the lengths and sequences of primers for particular applications is well known to those of ordinary skill in the art, as evidenced by the following reference that is incorporated by reference herein in its entirety: Dieffenbach, editor, PCR Primer: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Press, New York, 2003).

[0067] As used herein, the term “subject” refers to any living or non-living organism, including, but not limited to a human (e.g., a male human, female human, fetus, pregnant female, child, or the like), a non-human animal, a plant, a bacterium, a fungus or a protist. Any human or non-Attorney Docket No. HRG-027WO human animal can serve as a subject, including but not limited to mammal, reptile, avian, amphibian, fish, ungulate, ruminant, bovine (e.g., cattle), equine (e.g., horse), caprine and ovine (e.g., sheep, goat), swine (e.g., pig), camelid (e.g., camel, llama, alpaca), monkey, ape (e.g., gorilla, chimpanzee), ursid (e.g., bear), poultry, dog, cat, mouse, rat, fish, dolphin, whale and shark. In some embodiments, a subject is a male or female of any age (e.g., a man, a women or a child).

[0068] As used herein, “nucleoside” refers to a nucleobase linked to a sugar. The term “nucleoside” also includes a “modified nucleoside” which has independently, a modified sugar moiety and / or modified nucleobase. In various embodiments, a nucleoside refers to a xenonucleic acid, examples of which include a locked nucleic acid (LNA), hexitol nucleic acid (HNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), cyclohexenyl nucleic acid (CeNA), or peptide nucleic acid (PNA).

[0069] As used herein “selective enrichment of nucleic acid molecules” refers to the increased enrichment of nucleic acid molecules in relation to other nucleic acids. In various embodiments, selective enrichment refers to at least a fold enrichment of nucleic acid molecules relative to other nucleic acid molecules. Thus, in scenarios involving amplification and / or hybrid capture, selective enrichment of nucleic acid molecules does not require complete abatement or elimination of amplification and / or hybrid capture of other nucleic acid molecules. Rather, at least a fold increase in the amplification and / or hybrid capture of nucleic acid molecules is achieved in comparison to other nucleic acid molecules. In various embodiments, selective enrichment of nucleic acid molecules refers to at least a 2-fold increase, at least a 3 -fold increase, at least a 4-fold increase, at least a 5-fold increase, at least a 6-fold increase, at least a 7-fold increase, at least a 8-fold increase, at least a 9-fold increase, at least a 10-fold increase, at least a 15-fold increase, at least a 20-fold increase, at least a 25 fold increase, at least a 50-fold increase, at least a 100-fold increase, at least a 200-fold increase, at least a 500-fold increase, or at least a 1000-fold increase of the nucleic acid molecules relative to other nucleic acids.

[0070] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art.

[0071] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. SuchAttorney Docket No. HRG-027WO techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al. , Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); Coligan et al. , Short Protocols in Protein Science, John Wiley & Sons, NY (2003); Short Protocols in Molecular Biology (Wiley and Sons, 1999).

[0072] Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, biochemistry, immunology, molecular biology, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, and chemical analyses.

[0073] Throughout this specification and embodiments, the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0074] It is understood that wherever embodiments are described herein with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are also provided.

[0075] Any example(s) following the term “e.g.” or “for example” is not meant to be exhaustive or limiting.Attorney Docket No. HRG-027WO

[0076] Unless otherwise required by context, singular terms shall include pluralities, and plural terms shall include the singular.

[0077] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to be inclusive of the numbers defining the range and to encompass any and all subranges subsumed therein. For example, a stated range of “1 to 10” should be considered to include any and all subranges between (and inclusive of) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more, e.g., 1 to 6.1, and ending with a maximum value of 10 or less, e.g., 5.5 to 10.

[0078] Where aspects or embodiments of the disclosure are described in terms of a Markush group or other grouping of alternatives, the present disclosure encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group, but also the main group absent one or more of the group members. The present disclosure also envisages the explicit exclusion of one or more of any of the group members in an embodiment of the disclosure.

[0079] Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. The materials, methods, and examples are illustrative only and not intended to be limiting.Overview

[0080] Disclosed herein are methods for performing differential enrichment of nucleic acid molecules e.g., enriching for nucleic acid molecules comprising a target sequence. Such methods are useful for enriching for a signal in a sample, such as a signal informative for determining presence or absence of cancer in the sample. In various embodiments, methods disclosed herein are useful for enriching for hypermethylation sites by inhibiting or blocking non-methylated sites. For example, this enrichment technology can be used for methylation detection from bisulfite-converted DNA in qPCR, dPCR, hybridization capture, etc. The blockers outcompete the probe or primer by having a higher Tm, which allows for binding to targeted non-methylatedAttorney Docket No. HRG-027WODNA prior to the probe and / or primers, subsequently inhibiting hybridization of the probe or steps of PCR.

[0081] In various embodiments, methods disclosed herein are useful for enriching for variants of genomic regions. In various embodiments, methods disclosed herein are useful for enriching for genes with mutations of interest. In various embodiments, methods disclosed herein are useful for enriching for splice variants. In various embodiments, methods disclosed herein are useful for enriching for variations in transcripts. In various embodiments, methods disclosed herein are useful for enriching for variations in repetitive sequence elements.

[0082] In various embodiments, methods for performing differential enrichment of nucleic acid molecules involve providing a mixture of nucleic acid molecules comprising: a first set of nucleic acid molecules comprising a target sequence derived from a sequence comprising a wildtype gene sequence; and a second set of nucleic acid molecules comprising a candidate sequence derived from a sequence comprising a mutated gene sequence.

[0083] In various embodiments, methods for performing differential enrichment of nucleic acid molecules involve providing a mixture of nucleic acid molecules comprising: a first set of nucleic acid molecules comprising a target sequence derived from a sequence comprising a wildtype splice variant; and a second set of nucleic acid molecules comprising a candidate sequence derived from a sequence comprising an alternative splice variant.

[0084] In various embodiments, methods for performing differential enrichment of nucleic acid molecules involve providing a mixture of nucleic acid molecules comprising: a first set of nucleic acid molecules comprising a target sequence derived from a sequence comprising a repetitive element; and a second set of nucleic acid molecules comprising a candidate sequence derived from a sequence comprising mutations or rearrangements of the sequence in the repetitive element.

[0085] In various embodiments, methods for performing differential enrichment of nucleic acid molecules involve providing a mixture of nucleic acid molecules comprising: a first set of nucleic acid molecules comprising a target sequence derived from a sequence comprising one or more methylated CpG sites; and a second set of nucleic acid molecules comprising a candidate sequence derived from a sequence comprising one or more non-methylated CpG sites. Given the differentially methylated CpG sites in the first and second set of nucleic acid molecules, methods involve providing a blocker that selectively binds to the candidate sequence, or a portion thereof,Attorney Docket No. HRG-027WO of the second set of nucleic acid molecules, the blocker comprising a polymerase terminating blocker. The blocker may contain a sequence that is at least 90%, at least 95%, or 100% complementary to the candidate sequence, or a portion thereof of the second set of nucleic acid molecules. Additionally, given that the first set of nucleic acid molecules was derived from a sequence comprising one or more methylated CpG sites, the blocker contains a sequence that contains one or more mismatches relative to the target sequence of the first set of nucleic acid molecules. Thus, the blocker does not bind to the first set of nucleic acid molecules (or binds to the first set of nucleic acid molecules at a rate that is less than a rate at which the blocker binds to the candidate sequence, or portion thereof, of the second set of nucleic acid molecules).

[0086] Methods disclosed herein further comprise selectively enriching for the first set of nucleic acid molecules comprising the target sequence in comparison to the second set of nucleic acid molecules comprising the candidate sequence, wherein the blocker comprising a polymerase terminating blocker prevents or reduces enrichment of the second set of nucleic acid molecules comprising the candidate sequence. As one example, methods may involve providing a probe that is capable of hybridizing to one or both of the target sequence of the first set of nucleic acid molecules and the candidate sequence of the second set of nucleic acid molecules. However, the presence of the bound blocker may outcompete the binding of the probe to the candidate sequence, or portion thereof, of the second set of nucleic acid molecules. In contrast, given the lack of the blocker bound to the target sequence of the first set of nucleic acid molecules, the probe can readily hybridize with the target sequence, or portion thereof, of the first set of nucleic acid molecules. In various embodiments, the first set of nucleic acid molecules can be enriched using the probe (e.g., through hybrid capture of the probe). As another example, methods may involve providing primers (e.g., forward / reverse primers) for amplification. The presence of the bound blocker to the candidate sequence, or portion thereof, of the second set of nucleic acid molecules prevents or reduces amplification of the second set of nucleic acid molecules. In contrast, given the lack of the blocker bound to the target sequence of the first set of nucleic acid molecules, the amplification of the first set of nucleic acid molecules using the primers can readily occur. Methods further include detecting the selectively enriched first set of nucleic acid molecules.

[0087] In various embodiments, a blocker includes a sequence that shares at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at leastAttorney Docket No. HRG-027WO93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity with any one of SEQ ID NOs: 1-6.

[0088] In various embodiments, the disclosed blocker includes a polymerase terminating blocker, examples of which include dideoxycytidine (ddC), inverted dT, C3 spacer, and / or phosphorylation. In various embodiments, the polymerase terminating blocker is located on a 3’ end of the blocker, and therefore, example polymerase terminating blockers include 3’ ddC, 3’ ddG, 3’ ddA, 3’ ddT, 3’ inverted dT, 3’ C3 spacer, 3’ amino, and 3’ phosphorylation. In various embodiments, a blocker includes the polymerase terminating modification ddC. In various embodiments, a blocker includes the polymerase terminating modification C3. In various embodiments, a blocker includes a sequence of any one of SEQ ID NOs: 1-6.

[0089] Reference is now made to FIG. 1 , which shows an example flow diagram for differentially enriching for nucleic acid molecules, in accordance with an embodiment. As shown in FIG. 1, step 115 involves obtaining a sample (e.g., a sample from a subject). In various embodiments, a sample is any of a tissue sample, a blood sample, a stool sample, a urine sample, a mucous sample, or a saliva sample. In particular embodiments, a sample is a blood sample or a serum sample. In particular embodiments, a sample is a tissue sample. The sample can be obtained by the subject or by a third party, e.g., a medical professional. Examples of medical professionals include physicians, emergency medical technicians, nurses, first responders, psychologists, phlebotomists, medical physics personnel, nurse practitioners, surgeons, dentists, and any other medical professional as would be known to one skilled in the art. In various embodiments, the one or more samples can be obtained from the subject by a reference lab.

[0090] In various embodiments, the sample obtained from the subject is a liquid biopsy sample. In various embodiments, the liquid biopsy sample includes nucleic acid molecules. Example nucleic acid molecules include DNA or RNA. In particular embodiments, the nucleic acid molecules include cell-free DNA (cfDNA). In various embodiments, the cfDNA includes genomic sequences corresponding to CpG islands (CGIs) for which methylation states are informative for presence or absence of cancer. In various embodiments, the cfDNA can be derived from tumor cells and is referred to herein as circulating tumor DNA (ctDNA). In various embodiments, the nucleic acid molecules include a mixture of nucleic acid molecules that contain either methylated CpG sites or non-methylated CpG sites. For example, for a particularAttorney Docket No. HRG-027WO genomic region containing one or more CpG sites, the mixture of nucleic acid molecules includes a subset of nucleic acid molecules in which the one or more CpG sites are unmethylated and in a different subset of nucleic acid molecules in which the one or more CpG sites are partially or fully methylated.

[0091] Step 120 involves converting the nucleic acid molecules from the sample obtained from the subject. In various embodiments, converting the nucleic acid involves converting unmethylated nucleotides (e.g., cytosines) to another nucleotide (a “converted nucleotide,” as used herein). In various embodiments, methylated cytosines are protected from conversion (e.g., deamination) during the conversion step. Further details of performing conversion of nucleic acid molecules (e.g., step 120) are described herein.

[0092] Although not shown in FIG. 1 , in various embodiments, after conversion of nucleic acids, the converted nucleic acids undergo library construction. In various embodiments, converted nucleic acids can undergo end-repairing, tailing of 3’ ends, and / or addition of library or sequencing adapters. In various embodiments, converted nucleic acids can undergo biotinylation (e.g., addition of biotin moieties to converted nucleic acids). In various embodiments, barcodes can be incorporated into converted nucleic acids, thereby enabling subsequent sample demultiplexing (e.g., demultiplexing to identify sources of converted nucleic acids or demultiplexing to identify a common source from converted nucleic acids). In various embodiments, one or more washes and / or selections can be performed to remove unwanted DNA fragments, such as single stranded DNA fragments, excess adapters, and other molecules. In particular embodiments, a solid-phase reversible immobilization (SPRI) selection is performed. As used herein, a “nucleic acid template” refers to a nucleic acid derived from the converted nucleic acid (e.g., any of a nucleic acid derived from a converted nucleic acid that underwent library construction, end-repairing, addition of library or sequencing adapters, barcode addition, or any combination thereof).

[0093] Step 125 involves enriching for a subset of nucleic acid molecules. As shown in FIG. 1, step 125 can include substeps 130 and 140. Generally, enriching for a subset of nucleic acid molecules comprises providing one or more blockers comprising a polymerase terminating modification. The blocker binds to candidate sequences of converted nucleic acid molecules, or the complementary sequence thereof, the candidate sequences derived from sequences comprising one or more non-methylated CpG sites. For example, a candidate sequence mayAttorney Docket No. HRG-027WO contain nucleotides that were converted from corresponding non-methylated CpG sites and / or subsequently amplified. Thus, following conversion, a non-methylated CpG site would be converted to a “UG” sequence. Subsequent amplification may generate a corresponding “TG” sequence that is present in the candidate sequence, and “AC” in the complementary strand.

[0094] Specifically, step 130 involves providing a blocker that binds to a candidate sequence, or a portion thereof. In various embodiments, the blocker comprises between 10 and 50 nucleosides. In various embodiments, the blocker comprises between 11 and 45 nucleosides, between 12 and 40 nucleosides, between 13 and 35 nucleosides, between 15 and 35 nucleosides, or between 20 and 35 nucleosides. In various embodiments, the blocker comprises between 20 and 35 nucleosides. In various embodiments, the blocker comprises 20, 21, 22, 23, 24, 25, 26, 27, 38, 29, 30, 31, 32, 33, 34, or 35 nucleosides. In particular embodiments, the blocker comprises 25 nucleosides. In particular embodiments, the blocker comprises 26 nucleosides. In particular embodiments, the blocker comprises 26 nucleosides. In particular embodiments, the blocker comprises 27 nucleosides. In particular embodiments, the blocker comprises 28 nucleosides. In particular embodiments, the blocker comprises 29 nucleosides. In particular embodiments, the blocker comprises 30 nucleosides. In particular embodiments, the blocker comprises 31 nucleosides. In particular embodiments, the blocker comprises 32 nucleosides.

[0095] In various embodiments, the blocker comprises a polymerase terminating modification. Example polymerase terminating modification include 3’ ddC, 3’ ddG, 3’ ddA, 3’ ddT, 3’ inverted dT, 3 ’ C3 spacer, 3 ’ amino, and 3 ’ phosphorylation. In various embodiments, a blocker comprises the polymerase terminating modification ddC. In various embodiments, the blocker comprises the polymerase terminating modification C3.

[0096] In various embodiments, a blocker comprises a sequence that overlaps with the 3’ end of a forward primer or reverse primer overlaps by at least 3 nucleotides, at least 5 nucleotides, at least 7 nucleotides, at least 9 nucleotides, or at least 11 nucleotides. In some embodiments, the 3’ end of the primer overlaps with the sequence bound by the blocker by 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, or 11 nucleotides. In some embodiments, the 3’ end of the primer overlaps with the sequence the blocker binds to by 5 nucleotides. In some embodiments, the 3’ end of the primer overlaps with the sequence the blocker binds to by 6 nucleotides. In some embodiments, the 3’ end of the primer overlaps with the sequence theAttorney Docket No. HRG-027WO blocker binds to by 7 nucleotides. In some embodiments, the 3’ end of the primer overlaps with the sequence the blocker binds to by 8 nucleotides. In some embodiments, the 3’ end of the primer overlaps with the sequence the blocker binds to by 9 nucleotides. In some embodiments, the 3’ end of the primer overlaps with the sequence the blocker binds to by 10 nucleotides. In some embodiments, the 3 ’ end of the primer overlaps with the sequence the blocker binds to by 11 nucleotides.

[0097] In various embodiments, the blocker binds to a position complementary to a nucleotide derived from a cytosine of a CpG site. For example, the corresponding CpG site may have been unmethylated. Therefore, following conversion and / or amplification, the resulting nucleotides of the candidate sequence may be “TG” The blocker may comprise a nucleotide at a position complementary to the “T” (which is derived from the cytosine of the CpG site) or complementary to “A” (the complement of “T”). In various embodiments, the blocker binds to at least one CpG site. In various embodiments, the blocker binds to at least two CpG sites. In various embodiments, the blocker binds to at least three CpG sites.

[0098] In various embodiments, methods involve exposing the blocker and the candidate sequence to one or more temperatures to facilitate the binding between the blocker and the candidate sequence. In various embodiments, methods involve exposing the blocker and the candidate sequence to a temperature above 50°C, above 55°C, above 60°C, above 65°C, above 70°C, above 75°C, above 80°C, above 85°C, above 90°C, above 91 °C, above 92°C, above 93°C, above 94°C, or above 95°C to facilitate the binding between the blocker and the candidate sequence. In various embodiments, methods involve exposing the blocker and the candidate sequence to a temperature between 50°C and 65 °C to facilitate the binding between the blocker and the candidate sequence. In various embodiments, methods involve exposing the blocker and the candidate sequence to a temperature of about 50°C, about 51 °C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61 °C, about 62°C, about 63 °C, about 64°C, or about 65°C. In particular embodiments, methods involve exposing the blocker and the candidate sequence to a temperature of about 52°C. In particular embodiments, methods involve exposing the blocker and the candidate sequence to a temperature of about 56°C.

[0099] In various embodiments, the one or more temperatures comprises two different temperatures, wherein a first temperature is higher than a second temperature. In variousAttorney Docket No. HRG-027WO embodiments, the first temperature may be above 70°C, above 75°C, above 80°C, above 85°C, above 90°C, above 91°C, above 92°C, above 93°C, above 94°C, or above 95°C. In various embodiments, the first temperature is between about 70°C and 90 °C. In various embodiments, the first temperature is between about 70°C and 90 °C, between about 75°C and 85°C, or between about 75°C and 80°C. In various embodiments, the first temperature is about 75°C, about 76°C, about 77°C, about 78°C, about 79°C, or about 80°C. In various embodiments, the second temperature is between 50°C and 65°C, between 55°C and 62°C, between 58°C and 61 °C, or between 59°C and 60°C. In various embodiments, the second temperature is about 58°C, about 59°C, about 61°C, or about 61°C.

[0100] In various embodiments, methods further include providing a second blocker in addition to the first blocker. The second blocker can be provided simultaneously with the first blocker, or provided after the first blocker. Here, the second blocker binds to a different candidate sequence, or a portion thereof, of the second set of nucleic acid molecules. For example, the first blocker and the second blocker bind to different, non-overlapping candidate sequences, or portions thereof, of the second set of nucleic acid molecules. In these embodiments, the two blockers may bind to the different candidate sequences of the second set of nucleic acid molecules and can further improve the subsequent differential enrichment of nucleic acid molecules.

[0101] Returning to FIG. 1, step 140 involves selectively enriching for a first set of nucleic acid molecules comprising a target sequence (e.g., target sequence derived from one or more methylated CpG sites). Here, the first of nucleic acid molecules are selectively enriched in comparison to a second set of nucleic acid molecules comprising a candidate sequence (e.g., candidate sequence derived from one or more non-methylated CpG sites). Here, the presence of the blocker bound to the candidate sequence, or a portion thereof, prevents or reduces enrichment of the second set of nucleic acid molecules comprising the candidate sequence. For example, the blocker can prevent or reduce amplification of the second set of nucleic acid molecules comprising the candidate sequence. As another example, the blocker can outcompete binding of a primer, thereby preventing subsequent detection methods (e.g., qPCR) that rely on the primer for amplification of the sequence.

[0102] In various embodiments, selectively enriching for a first set of nucleic acid molecules comprises providing a probe capable of hybridizing to the target sequence, or aAttorney Docket No. HRG-027WO portion thereof, of the first set of nucleic acid molecules. In various embodiments, the probe can be provided simultaneously with the blocker or can be provided after the blocker. Here, the probe hybridizes with the target sequence, or a portion thereof of the first set of nucleic acid molecules, thereby enabling subsequent enrichment of the first set of nucleic acid molecules. For example, a subsequent enrichment can involve performing a hybrid capture. In hybrid capture, solid supports, such as solid beads, can capture the probe, thereby enriching for the first set of nucleic acid molecules bound to the probe. In various embodiments, solid beads can be streptavidin coated beads that capture biotinylated probes that are bound to the first set of nucleic acid molecules.

[0103] In various embodiments, a primer is unable to hybridize with the candidate sequence of a second set of nucleic acids due to presence of a bound blocker. In various embodiments, the primer is complementary to a portion of the candidate sequence that is bound by the blocker. For example, the primer may be complementary to at least one nucleoside, at least two nucleosides, at least three nucleosides, at least four nucleosides, at least five nucleosides, at least six nucleosides, at least seven nucleosides, at least eight nucleosides, at least nine nucleosides, at least ten nucleosides, at least eleven nucleosides, at least twelve nucleosides, at least thirteen nucleosides, at least fourteen nucleosides, at least fifteen nucleosides, at least sixteen nucleosides, at least seventeen nucleosides, at least eighteen nucleosides, at least nineteen nucleosides, or at least twenty nucleosides that are bound by the blocker. The blocker may outcompete the primer for binding to the candidate sequence, or a portion thereof. The blocker prevents the primer from binding to the candidate sequence, or a portion thereof, of the second set of nucleic acids and therefore prevents subsequent enrichment of the second set of nucleic acids.

[0104] In various embodiments, a blocker outcompetes the primer for binding to the candidate sequence if the blocker, when bound to the candidate sequence, achieves a threshold melting temperature. In various embodiments, the threshold melting temperature is at least 60°C, 65°C, at least 66°C, at least 67°C, at least 67°C, at least 69°C, at least 70°C, at least 71°C, at least 72°C, at least 73°C, at least 74°C, or at least 75°C. In particular embodiments, the threshold melting temperature is about 65°C. In various embodiments, the blocker, when bound to the candidate sequences, increases the melting temperature to 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, or 70°C. In various embodiments, a blocker outcompetes the primer for binding toAttorney Docket No. HRG-027WO the candidate sequence if the blocker, when bound to the candidate sequence, achieves a melting temperature that is at least 1°C higher, at least 2°C higher, at least 3°C higher, at least 4°C higher, at least 5°C higher, at least 6°C higher, at least 7°C higher, at least 8°C higher, at least 9°C higher, at least 10°C higher, at least 11°C higher, at least 12°C higher, at least 13 °C higher, at least 14°C higher, or at least 15°C higher than a melting temperature of the candidate sequence bound to the primer. In particular embodiments, a blocker outcompetes the primer for binding to the candidate sequence if the blocker, when bound to the candidate sequence, achieves a melting temperature that is at least 5°C higher than a melting temperature of the candidate sequence bound to the primer.

[0105] In various embodiments, selectively enriching for a first set of nucleic acid molecules comprises providing primers for performing nucleic acid amplification. In various embodiments, primers can be provided simultaneously with the blocker or can be provided after the blocker. In various embodiments, the primers comprise a primer pair (e.g., a forward primer and a reverse primer). In various embodiments, the primers may be capable of hybridizing to complementary sequences on the first set of nucleic acid molecules. Thus, nucleic acid amplification of the first set of nucleic acid molecules can occur. In various embodiments, a nucleobase of the primer when hybridized to a nucleic acid of a second set of nucleic acid molecules is immediately adjacent to the blocker that is bound to the candidate sequence of the second set of nucleic acid molecules. The presence of the blocker may prevent subsequent amplification of the second set of nucleic acid molecules as the blocker prevents extension along the nucleic acid molecule.

[0106] In some embodiments, the primers are incapable of hybridizing to complementary sequences of the second set of nucleic acid molecules due to the presence of the bound blocker to a candidate sequence of the second set of nucleic acid molecules. For example, the blocker may be bound to a portion of the second set of nucleic acid molecules that is complementary to a sequence of a primer. Thus, as the primers are unable to bind to complementary sequences of the second set of nucleic acid molecules, performing nucleic acid amplification does not amplify the second set of nucleic acid molecules. In various embodiments, the blocker and a primer may both be complementary to an overlapping sequence of at least 1 nucleoside. In various embodiments, the blocker and a primer may both be complementary to an overlapping sequence of at least 2 nucleosides, at least 3 nucleosides, at least 4 nucleosides, at least 5 nucleosides, atAttorney Docket No. HRG-027WO least 6 nucleosides, at least 7 nucleosides, at least 8 nucleosides, at least 9 nucleosides, at least 10 nucleosides, at least 11 nucleosides, at least 12 nucleosides, at least 13 nucleosides, at least 14 nucleosides, or at least 15 nucleosides.

[0107] In various embodiments, selectively enriching for a first set of nucleic acid molecules comprises providing both 1) a probe capable of hybridizing to the target sequence, or a portion thereof, of the first set of nucleic acid molecules and 2) primers for performing nucleic acid amplification. In various embodiments, the probe and primers can each be provided simultaneously with the blocker or can be provided after the blocker.

[0108] Although not shown in FIG. 1, in various embodiments, after step 140, the enriched nucleic acids undergo one or more washes and / or selections to remove unwanted DNA fragments, such as single stranded DNA fragments, excess primers, excess adapters, and other molecules. In particular embodiments, a solid-phase reversible immobilization (SPRI) selection is performed.

[0109] Step 150 involves detecting the selectively enriched first set of nucleic acid. In various embodiments, detecting the selectively enriched nucleic acids involves performing sequencing to determine the sequences of the first set of nucleic acids. In various embodiments, sequencing data can be demultiplexed e.g., using barcode sequences. In various embodiments, sequencing data can be aligned to a reference genome and / or trimmed. In various embodiments, sequencing data can be further analyzed to determine the enriched signal in the sample e.g., for determining presence or absence of cancer in the sample. In various embodiments, detecting the selectively enriched nucleic acids involves quantifying a signal from the first set of nucleic acids. For example, the signal may be a fluorescent signal. Thus, quantifying the fluorescent signal can be informative for determining a total quantity of the first set of nucleic acid molecules (or nucleic acids, such as amplicons, derived from the first set of nucleic acid molecules. Detecting the selectively enriched nucleic acids by quantifying a signal from the first set of nucleic acids can be performed e.g., when performing quantitative PCR.Methods for Enriching Target Nucleic Acid Sequences a. Exemplary Methods

[0110] As disclosed herein in reference to FIG. 1, methods can involve step 125 for enriching a first set of nucleic acid molecules e.g., nucleic acid molecules comprising targetAttorney Docket No. HRG-027WO nucleic acid sequences. Methods for enriching the first set of nucleic acid molecules is further described in reference to FIGs. 3A-3D, which depict diagrams involving provision of blockers and / or probe / primers, in accordance with various embodiments.

[0111] Beginning with FIG. 3A, it depicts a diagram involving blockers, in accordance with an embodiment. Step A begins with converted nucleic acids 410, including nucleic acid molecule 415A and nucleic acid molecule 415B. Step A shows the converted nucleic acids 410 after having performed step 120 shown in FIG. 1. Here, nucleic acid molecule 415A may include a target sequence 405 that is derived from a sequence comprising one or more methylated CpG sites. FIG. 3A shows two genomic sites (labeled as “m”) that are derived from corresponding CpG sites 402 that were previously methylated. For example, the prior CpG sites may have a sequence of “CG” where the cytosine was methylated. Following conversion, the “CG” remains and the target sequence 405 can include a “CG,” or complement thereof, at each site corresponding to a CpG site.

[0112] Additionally, nucleic acid molecule 415B includes a candidate sequence 408 that is derived from a sequence comprising one or more non-methylated CpG sites. Specifically, FIG. 3A shows two genomic sites that are derived from corresponding CpG sites 404 that were previously unmethylated. For example, the prior CpG sites may have a sequence of “CG” where the cytosine was non-methylated. Following conversion, the “CG” is converted to “UG.” Thus, the candidate sequence 408 can include a “TG,” or complement thereof, at each site corresponding to a CpG site.

[0113] Following step A, one or more blockers 420 are introduced to the converted nucleic acids 410. The one or more blockers bind to a candidate sequence 408, or a portion thereof, of nucleic acid molecule 415B. Although step B of FIG. 3 A shows a single blocker 420 binding to candidate sequence 408, other embodiments can include multiple blockers 420 binding to different candidate sequences on a nucleic acid molecule 415B.

[0114] The one or more blockers 420 include terminating modifications. For example, as shown in FIG. 3 A, the blockers 420 may include terminating modifications (421, indicated by “X”) located at the 3’ end of the blockers 420. The terminating modification “X” can be a nucleoside e.g., a ddC or inverted dT, or it can be a modification e.g., 3’ C3 spacer, 3’ amino, and 3’ phosphorylation.Attorney Docket No. HRG-027WO

[0115] In various embodiments, a blocker 420 does not bind, or minimally binds, to target sequence 405. Given the mismatches between the sequence of the blocker 420 and the nucleotides of the target sequence 405 that correspond to methylated CpG sites 402, the blocker 420 may fail to hybridize, or hybridizes to a lesser extent, with the target sequence 405. In various embodiments, less than 50% of nucleic acid molecule 415A (e.g., first set of nucleic acid molecules that are derived from sequences with one or more methylated CpG sites) are bound to a blocker 420. In various embodiments, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% of nucleic acid molecule 415A are bound to a blocker 420.

[0116] Next, one or both of probes and / or primers are provided. FIGs. 3B-3D show three separate embodiments in which only probes are provided (e.g., Step C. 1 in FIG. 3B), only primers are provided (Step C. 2 in FIG. 3C), or probes and primers are provided (Step C. 3 in FIG. 3D).

[0117] Reference is first made to FIG. 3B, which depicts a diagram involving provision of probes 430, in accordance with a first embodiment. The probe 430 is capable of hybridizing to the target sequence 405, or a portion thereof, of the nucleic acid molecule 415A. In various embodiments, the probe 430 comprises a sequence that is at least 90% complementary, at least 95% complementary, at least 96% complementary, at least 97% complementary, at least 98% complementary, at least 99% complementary, or 100% complementary to the target sequence, or a portion thereof. In various embodiments, the probe 430 is also capable of hybridizing to the candidate sequence 408, albeit to a lesser extent than hybridizing to the target sequence 405. However, the presence of the blocker 420 bound to the candidate sequence 408, with a terminating modification 421, prevents or reduces the binding of the probe 430 to the candidate sequence 408. In various embodiments, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1% of nucleic acid molecule 415B are bound to a probe 430.Attorney Docket No. HRG-027WO

[0118] Given that the probe 430 is bound to nucleic acid molecule 415 A and not bound to nucleic acid molecule 415B, subsequent enrichment can be performed to enrich for nucleic acid molecule 415A using the bound probe 430. For example, methods can involve performing hybrid capture e.g., using a solid support to capture the probe 430 bound to the target sequence 405 of the nucleic acid molecule 415 A. Thus, the nucleic acid molecule 415A can be enriched through hybrid capture while eliminating or reducing the quantity of nucleic acid molecule 415B.

[0119] Reference is next made to FIG. 3C, which depicts a diagram involving provision of primers, in accordance with a second embodiment. The primers 435 may include a primer set e.g., forward and reverse primers. As shown in FIG. 3C, the primers 435A and 435B may be capable of hybridizing to complementary sequences on the nucleic acid molecule 415A and the nucleic acid molecule 415B. In this scenario, the presence of the blocker 420, with a terminating modification 421, bound to a candidate sequence 408 of nucleic acid molecule 415B substantially affects the binding of the primer 435A to the nucleic acid molecule 415B. In another scenario, the presence of the blocker 420 bound to a candidate sequence 408 of nucleic acid molecule 415B substantially affects the binding of the primer 435B to the nucleic acid molecule 415B.

[0120] Given the lack of a bound blocker 420 on the nucleic acid molecule 415 A, selective nucleic acid amplification of nucleic acid molecule 415A can occur. For example, nucleic acid amplification can be initiated using the bound primers 435 A and 435B, thereby generating amplicons of nucleic acid molecule 415 A. In contrast, the presence of the bound blocker 420, with a terminating modification 421, on the nucleic acid molecule 415B may prevent or reduce the amplification of nucleic acid molecule 415B. Specifically, the blocker 420 may prevent extension of a strand initiated from primer 435 A. Thus, the resulting amplicons derived from nucleic acid molecule 415B can be prevented or reduced.

[0121] Although FIG. 3C shows the blocker 420 as binding to a candidate sequence 408 located overlapping with a sequence bound by a primer e.g., primer 435A. The amount of overlap between the blocker 420 and the primer 435 A may increase how effective the blocker is in preventing or reducing amplification of the nucleic acid molecule 415B.

[0122] Reference is next made to FIG. 3D, which depicts a diagram involving provision of both probes and primers, in accordance with a third embodiment. Although the probe 430 andAttorney Docket No. HRG-027WO primers 435 are shown to be provided simultaneously to nucleic acid molecules 415 A and nucleic acid molecule 415B, in various embodiments, they are provided in a successive order.

[0123] The probe 430 is capable of hybridizing to the target sequence 405, or a portion thereof, of the nucleic acid molecule 415A. The primers 435 may include a primer set e.g., forward and reverse primers. The primers 435 A and 435B may be capable of hybridizing to complementary sequences on the nucleic acid molecule 415 A and the nucleic acid molecule 415B.

[0124] Probe 430 is bound to nucleic acid molecule 415A, and may bind to nucleic acid molecule 415B after the blocker is bound, subsequent enrichment can be performed to enrich for nucleic acid molecule 415 A using the bound probe 430. For example, methods can involve performing hybrid capture e.g., using a solid support to capture the probe 430 bound to the target sequence 405 of the nucleic acid molecule 415A. Thus, the nucleic acid molecule 415A can be enriched through hybrid capture while eliminating or reducing the quantity of nucleic acid molecule 415B. Additionally the nucleic acid molecule 415A can be detected by fluorescence while eliminating or reducing detection of nucleic acid molecule 415B. Nucleic acid amplification can be initiated using the bound primers 435 A and 435B, thereby generating amplicons of nucleic acid molecule 415 A. In contrast, the presence of the bound blocker 420 on the nucleic acid molecule 415B may prevent or reduce the amplification of nucleic acid molecule 415B. Specifically, the blocker 420 may prevent primer 435A from binding nucleic acid molecule 415B or the extension of a strand initiated from primer 435 A. Thus, the resulting amplicons derived from nucleic acid molecule 415B can be prevented or reduced. Thus, in comparison to the scenarios shown in FIGs. 4B and 4C, FIG. 3D may achieve improved differential enrichment of nucleic acid molecule 415A by both 1) preventing probe 430 from binding to nucleic acid molecule 415B and 2) preventing or reducing amplification of nucleic acid molecule 415B using primers 435. b. Example Designs of Sequences

[0125] As disclosed herein, a blocker may contain a sequence that is at least 90%, at least 95%, or 100% complementary to a candidate sequence, or a portion thereof. In various embodiments, a blocker may contain a sequence that is at least 90%, at least 91%, at least 92%,Attorney Docket No. HRG-027WO at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or 98% complementary to a target sequence, or a portion thereof. In various embodiments, a blocker may be designed to be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or 98% complementary to a target sequence or a candidate sequences that includes one or more CpG sites within a region disclosed in Table 1 or Table 2. In various embodiments, a blocker may be designed to be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or 98% complementary to a target sequence or a candidate sequences that includes two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more CpG sites within a region disclosed in Table 1 or Table 2. In various embodiments, a blocker may be designed to be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98% complementary to a target sequence or a candidate sequences that includes two, three, four, five, six, seven, eight, nine, or ten CpG sites within a region disclosed in Table 1 or Table 2. In various embodiments, a blocker may be designed to be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98% complementary to a target sequence or a candidate sequences that includes five sequential CpG sites within a region disclosed in Table 1 or Table 2. In various embodiments, a blocker is designed to be 96% complementary to a target sequence or a candidate sequences that includes five sequential CpG sites within a region disclosed in Table 1 or Table 2.

[0126] In various embodiments, a blocker may be designed to be complementary to a candidate sequence that contains X CpG sites, where the X CpG sites are fully unmethylated. In various embodiments, a blocker may be designed to be complementary to a candidate sequence that contains X CpG sites, where the X CpG sites are fully methylated. In various embodiments, a blocker may be designed to be complementary to a candidate sequence that contains X CpG sites, where at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the X CpG sites are unmethylated. As referred to herein, the term “K*n#” refers to a sequence having CpG sites, whereof the CpG sites that are methylated. Therefore, the term “K5n5” refers to a sequence including 5 CpG sites in which 5 of the CpG sites are methylated. As another example, the term “K6n5” refers to a sequence including 6 CpG sites in which 5 of the CpG sites are methylated. In various embodiments, can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In various embodiments,can be any of 1, 2, 3, 4,Attorney Docket No. HRG-027WO5, 6, 7, 8, 9, or 10. In some embodiments, a candidate sequence is less than 200 bp, less than 150 bp, less than 100 bp, less than 75 bp, less than 50 bp in length, or less than 35 bp in length. In some embodiments, a candidate sequence is less than 150 bp in length. In some embodiments, a candidate sequence is less than 100 bp in length. In some embodiments, a candidate sequence is less than 75 bp in length. In some embodiments, a candidate sequence is less than 50 bp in length. In some embodiments, a candidate sequence is less than 35 bp in length.

[0127] In various embodiments, a blocker may be designed to be fully complementary to a candidate sequence derived from a “K*n#” sequence. For example, a blocker may be designed to be complementary to a candidate sequence derived from a K5n0 sequence (e.g., fully unmethylated sequence of 5 CpG sites). Thus, as each of the CpG sites of the K5n0 sequence is unmethylated, the resulting sequence corresponding to each CpG site may be “TG,” or a complement thereof. Thus, the blocker may be designed to have a sequence that is complementary to each of the five “TG,” or complement thereof.

[0128] In various embodiments, a blocker may be designed to be complementary to a candidate sequence derived from any of a KlnO sequence, a K2n0 sequence, a K3n0 sequence, a K4n0 sequence, a K5n0 sequence, a K6n0 sequence, a K7n0 sequence, a K8n0 sequence, a K9n0 sequence, or a KlOnO sequence. In various embodiments, a blocker may be designed to be complementary to a candidate sequence derived from any of a K2nl sequence, a K3nl sequence, a K4nl sequence, a K5nl sequence, a K6nl sequence, a K7nl sequence, a K8nl sequence, a K9nl sequence, a KlOnl sequence, a K3n2 sequence, a K4n2 sequence, a K5n2 sequence, a K6n2 sequence, a K7n2 sequence, a K8n2 sequence, a K9n2 sequence, a K10n2 sequence, a K4n3 sequence, a K5n3 sequence, a K6n3 sequence, a K7n3 sequence, a K8n3 sequence, a K9n3 sequence, a K10n3 sequence, a K5n4 sequence, a K6n4 sequence, a K7n4 sequence, a K8n4 sequence, a K9n4 sequence, a K10n4 sequence, a K6n5 sequence, a K7n5 sequence, a K8n5 sequence, a K9n5 sequence, a K10n5 sequence, a K7n6 sequence, a K8n6 sequence, a K9n6 sequence, a K10n6 sequence, a K8n7 sequence, a K9n7 sequence, a K10n7 sequence, a K9n8 sequence, a K10n8 sequence, or a K10n9 sequence.

[0129] As disclosed herein, a probe may contain a sequence that is at least 90% complementary, at least 95% complementary, at least 96% complementary, at least 97% complementary, at least 98% complementary, at least 99% complementary, or 100% complementary to the target sequence, or a portion thereof. In various embodiments, a probeAttorney Docket No. HRG-027WO may be designed to be at least 90%, at least 95%, or 100% complementary to a target sequence that includes one or more CpG sites within a region disclosed in Table 1 or Table 2. In various embodiments, a probe may be designed to be at least 90%, at least 95%, or 100% complementary to a target sequence that includes two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more CpG sites within a region disclosed in Table 1 or Table 2. In various embodiments, a probe may be designed to be at least 90%, at least 95%, or 100% complementary to a target sequence that includes two, three, four, five, six, seven, eight, nine, or ten CpG sites within a region disclosed in Table 1 or Table 2. In various embodiments, a probe may be designed to be at least 90%, at least 95%, or 100% complementary to a target sequence that includes five sequential CpG sites within a region disclosed in Table 1 or Table 2. In various embodiments, a probe is designed to be 100% complementary to a target sequence that includes five sequential CpG sites within a region disclosed in Table 1 or Table 2.

[0130] In various embodiments, a probe may be designed to be complementary to a target sequence that contains X CpG sites, where the X CpG sites are fully methylated. In various embodiments, a probe may be designed to be complementary to a target sequence that contains X CpG sites, where the X CpG sites are fully methylated. In various embodiments, a probe may be designed to be complementary to a target sequence that contains X CpG sites, where at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the X CpG sites are methylated.

[0131] In various embodiments, a probe may be designed to be fully complementary to a target sequence derived from a “K*n#” sequence. For example, a probe may be designed to be complementary to a target sequence derived from a K5n5 sequence (e.g., fully methylated sequence of 5 CpG sites). Thus, as each of the CpG sites of the K5n5 sequence is methylated, the resulting sequence corresponding to each CpG site may be “CG,” or a complement thereof. Thus, the probe may be designed to have a sequence that is complementary to each of the five “CG,” or complement thereof.

[0132] In various embodiments, a probe may be designed to be complementary to a target sequence derived from any of a Klnl sequence, a K2n2 sequence, a K3n3 sequence, a K4n4 sequence, a K5n5 sequence, a K6n6 sequence, a K7n7 sequence, a K8n8 sequence, a K9n9 sequence, or a KlOnlO sequence. In various embodiments, a probe may be designed to beAttorney Docket No. HRG-027WO complementary to a target sequence derived from any of a K2nl sequence, a K3nl sequence, a K4nl sequence, a K5nl sequence, a K6nl sequence, a K7nl sequence, a K8nl sequence, a K9nl sequence, a KlOnl sequence, a K3n2 sequence, a K4n2 sequence, a K5n2 sequence, a K6n2 sequence, a K7n2 sequence, a K8n2 sequence, a K9n2 sequence, a K10n2 sequence, a K4n3 sequence, a K5n3 sequence, a K6n3 sequence, a K7n3 sequence, a K8n3 sequence, a K9n3 sequence, a K10n3 sequence, a K5n4 sequence, a K6n4 sequence, a K7n4 sequence, a K8n4 sequence, a K9n4 sequence, a K10n4 sequence, a K6n5 sequence, a K7n5 sequence, a K8n5 sequence, a K9n5 sequence, a K10n5 sequence, a K7n6 sequence, a K8n6 sequence, a K9n6 sequence, a K10n6 sequence, a K8n7 sequence, a K9n7 sequence, a K10n7 sequence, a K9n8 sequence, a K10n8 sequence, or a K10n9 sequence. c. Enrichment Steps

[0133] In certain embodiments, the target sequence or a subset of target sequences in the nucleic acid can be enriched using one or more additional enrichment steps. The one or more additional enrichments steps can be performed using any enrichment method known in the art. Non-limiting examples include hybrid capture, use of DNA-binding proteins to enrich a target sequence or a subset of target sequences, and nucleic acid amplification (e.g., polymerase chain reaction). In various embodiments, an enrichment step involves performing one or more of PCR, RT-PCR, qPCR, digital PCR, nicking endonuclease amplification (NEAR), transcription- mediated amplification (TMA), loop-mediated isothermal amplification (LAMP), helicasedependent amplification (HAD), strand displacement amplification (SDA), sequencing library PCR, hybrid capture, microarrays, next generation sequencing (NGS), mass spectrometry, or hybrid capture PCR. In various embodiments, the additional enrichment step involves performing one or more of PCR or RT-PCR. In various embodiments, the additional enrichment step involves performing indexing PCR amplification e.g., using library adapters (e.g., P5 / P7 adapters). Thus, selective amplification nucleic acids with hybridized primer sequences can be performed using the library adapters. In contrast, nucleic acids in which primer sequences are incapable of hybridizing with do not undergo PCR amplification.

[0134] One or more additional enrichment steps can be performed before or after the blocking of the unmethylated nucleic acids, as described herein. For example, in certain embodiments, the method comprises a first step of depleting a first subset of nucleic acids (e.g.,Attorney Docket No. HRG-027WO unmethylated nucleic acids or converted nucleic acids derived from unmethylated nucleic acids), thereby leaving a second subset of nucleic acids (e.g., methylated nucleic acids or converted nucleic acids derived from methylated nucleic acids). The method comprises a second step of subjecting nucleic acid sequences comprising the target sequence to one or more additional enrichment steps to enrich for at least a subset of the target sequences.

[0135] In certain embodiments, the method comprises a first step of subjecting a plurality of nucleic acid molecules that include target sequences to an enrichment step to enrich for the target sequences. The method further comprises a second step of subjecting the plurality of nucleic acid molecules to the depletion method disclosed herein, which depletes a first subset of nucleic acids (e.g., unmethylated nucleic acids or converted nucleic acids derived from unmethylated nucleic acids) thereby leaving a second subset of nucleic acids (e.g., methylated nucleic acids or converted nucleic acids derived from methylated nucleic acids).

[0136] In certain embodiments, a target sequence or a subset of target sequences in the nucleic acid can be enriched by subjecting the nucleic acid comprising the target sequence or the subset of target sequences to hybrid capture. In hybrid capture, labeled (e.g., biotinylated) capture probes that can bind to one or more target sequences or subsets of target sequences are exposed to the nucleic acid comprising the one or more target sequences. The capture probes are specific to a sequence of interest, for example, a methylation pattern of interest that can be detected as a bisulfite-converted epitype. Examples of such hybrid capture probe sets include the KAPA HyperPrep KAPA HyperCap Workflow with HyperChoice Probes, Twist Bioscience Twist Fast Hybridization Custom Target Enrichment Panel, Integrated DNA technologies xGen Custom Hybridization Capture Panel, and SeqCAP Epi Enrichment System from Roche Diagnostics (Pleasanton, CA).

[0137] In various embodiments, the capture probe is specific for a sequence of interest of a probe to enrich for nucleic acid molecules that are bound by the probe. For example, as disclosed herein, a probe (such as probe 430 described in FIGs. 3B and 3C) may bind to a target sequence of a nucleic acid molecule (e.g., a methylated nucleic acid molecule). Therefore, when performing hybrid capture, the capture probe may bind to the probe and enrich for the target sequence of a nucleic acid molecule (e.g., a methylated nucleic acid molecule).Attorney Docket No. HRG-027WOExample Nucleic Acids and Methods for Converting Nucleic Acids

[0138] As discussed herein, step 120 in FIG. 1 involves converting nucleic acid molecules from the obtained sample. In various embodiments, converting nucleic acid molecules includes treating the nucleic acid molecules to capture methylation modifications. In various embodiments, converting nucleic acid molecules involves converting one or more unmethylated nucleotides (e.g., cytosines) to another nucleotide (a “converted nucleotide,” as used herein), e.g., using chemical or enzymatic means. In certain embodiments, one or more unmethylated cytosines are converted to a nucleotide that pairs with adenine (e.g., the unmethylated cytosine may be converted to uracil). In certain embodiments, one or more unmethylated adenines are converted to a base that pairs with cytosine (e.g., the unmethylated adenine may be converted to inosine (I)). In certain embodiments, one or more unmethylated cytosines (e.g., a 5- methylcytosine (5mC)) is converted to a thymine, which pairs with adenine. In certain embodiments, methylated cytosines are protected from conversion (e.g., deamination) during the conversion step.

[0139] After a nucleic acid has been treated to convert unmethylated, or, in some cases, methylated nucleotides, into another nucleotide, the nucleic acid may be amplified. During amplification, the converted nucleotide pairs with its complementary nucleotide, and in the next round of amplification, the complementary nucleotide pairs with a replacement nucleotide. For example, following the conversion of an unmethylated cytosine to a uracil, the nucleic acid may be amplified such that an adenine pairs with the uracil in the first round of replication, and in the second round of replication, the adenine pairs with a thymine. Accordingly, the thymine replaces the uracil in the original nucleic acid sequence, and is referred to herein as a “replacement nucleotide.”

[0140] In certain aspects, conversion of the nucleic acids involves selectively deaminating nucleotides. FIG. 2A depicts an example conversion of nucleic acids, in accordance with an embodiment. Selective deamination refers to a process in which unmethylated cytosine residues are selectively deaminated over methylated cytosine (5-methylcytosine) residues. In certain embodiments, deamination of cytosine forms uracil, effectively inducing a C to T point mutation to allow for detection of methylated cytosines. Methods of deaminating cytosine are known in the art, and include chemical conversion (e.g., bisulfite conversion) and enzymatic conversion. In certain embodiments, the enzymatic conversion comprises subjecting the nucleicAttorney Docket No. HRG-027WO acid to TET2, which oxidizes methylated cytosines, thereby protecting them, and subsequent exposure to APOBEC, which converts unprotected (i.e., unmethylated) cytosines to uracils.

[0141] In some embodiments, the conversion, for example, bisulfite conversion or enzymatic conversion, uses commercially available kits. Bisulfite conversion can be performed using commercially available technologies, such as EZ DNA Methylation-Gold, EZ DNAMethylation-Direct or an EZ DNAMethylation-Lighting kit (Zymo Research Corp (Irvine, California)) or EpiTect Fast available from Qiagen (Germantown, MD). In another example a kit such as APOBECSeq (NEBiolabs) or OneStep qMethyl-PCR Kit (Zymo Research Corp (Irvine, California)) is used. a. Source of Nucleic Acids

[0142] Nucleic acids used in the methods described herein can be derived from any source, such as a sample taken from the environment or from a subject (e.g., a human subject). A biological sample can be treated to physically disrupt tissue or cell structure (e.g., centrifugation and / or cell lysis), thus releasing intracellular components into a solution which can further contain enzymes, buffers, salts, detergents, and the like which can be used to prepare the sample for analysis. A biological sample can take any of a variety of forms, such as a liquid biopsy (e.g., blood, urine, stool, saliva, or mucous), or a tissue biopsy, or other solid biopsy. Examples of biological samples include, but are not limited to, blood, whole blood, plasma, serum, urine, cerebrospinal fluid, fecal, saliva, sweat, tears, pleural fluid, pericardial fluid, or peritoneal fluid of the subject. A biological sample can include any tissue or material derived from a living or dead subject. A biological sample can be a cell-free sample. A sample can be a liquid sample or a solid sample (e.g., a cell or tissue sample). A biological sample can be a bodily fluid, such as blood, plasma, serum, urine, vaginal fluid, fluid from a hydrocele (e.g., of the testis), vaginal flushing fluids, pleural fluid, ascitic fluid, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchoalveolar lavage fluid, discharge fluid from the nipple, aspiration fluid from different parts of the body (e.g., thyroid, breast), etc.

[0143] The nucleic acid can be of any composition form, such as deoxyribonucleic acid (DNA, e.g., complementary DNA (cDNA), genomic DNA (gDNA) and the like), and / or DNA analogs (e.g., containing base analogs, sugar analogs and / or a non-native backbone and the like), and / or ribonucleic acid (RNA) and / or RNA analogs, all of which can be in single- or doubleAttorney Docket No. HRG-027WO stranded form. In certain embodiments, single-stranded nucleic acids can be made double stranded prior to cutting with an enzyme. Unless otherwise limited, a nucleic acid can comprise known analogs of natural nucleotides, some of which can function in a similar manner as naturally occurring nucleotides. A nucleic acid can be in any form useful for conducting processes herein (e.g., linear, circular, supercoiled, single-stranded, double-stranded and the like). A nucleic acid in some embodiments can be from a single chromosome or fragment thereof (e.g., a nucleic acid sample may be from one chromosome of a sample obtained from a diploid organism). In certain embodiments nucleic acids comprise nucleosomes, fragments or parts of nucleosomes or nucleosome-like structures. Nucleic acids can comprise protein (e.g., histones, DNA binding proteins, and the like). Nucleic acids analyzed by processes described herein can be substantially isolated and are not substantially associated with protein or other molecules. Nucleic acids can also include derivatives, variants and analogs of DNA synthesized, replicated or amplified from single-stranded (“sense” or “antisense,” “plus” strand or “minus” strand, “forward” reading frame or “reverse” reading frame) and double-stranded polynucleotides. Deoxyribonucleotides can include deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine. A nucleic acid may be prepared using a nucleic acid obtained from a subject as a template.

[0144] In certain embodiments, the nucleic acid is a cell-free nucleic acid, which can be found in bodily fluids such as blood, whole blood, plasma, serum, urine, cerebrospinal fluid, fecal, saliva, sweat, sweat, tears, pleural fluid, pericardial fluid, or peritoneal fluid of a subject. In certain embodiments, a plasma sample can be used directly in the methods disclosed herein (for example, in the cutting step), without prior purification or isolation of nucleic acids in the plasma. Cell-free nucleic acids originate from one or more healthy cells and / or from one or more cancer cells, or from non-human sources such bacteria, fungi, viruses. Examples of the cell-free nucleic acids include but are not limited to cell-free DNA (“cfDNA”), including mitochondrial DNA or genomic DNA, and cell-free RNA. In certain embodiments herein, instruments for assessing the quality of the cell-free nucleic acids, such as the TapeStation System from Agilent Technologies (Santa Clara, CA) can be used. Concentrating low-abundance cfDNA can be accomplished, for example using a Qubit Fluorometer from Thermofisher Scientific (Waltham, MA).Attorney Docket No. HRG-027WO

[0145] In various embodiments, the majority of DNA in a biological sample that has been enriched for cell-free DNA (e.g., a plasma sample obtained via a centrifugation protocol) can be cell-free e.g., greater than 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the DNA can be cell-free).

[0146] A methylated nucleic acid is a nucleic acid having a modification in which a hydrogen atom on the pyrimidine ring of a cytosine base is converted to a methyl group, forming 5-methylcytosine. Methylation can occur at dinucleotides of cytosine and guanine referred to herein as “CpG sites,” which can be a target for enrichment. Methylation of cytosine can occur in cytosines in other sequence contexts, for example, 5'-CHG-3' and 5'-CHH-3', where H is adenine, cytosine or thymine. Cytosine methylation can also be in the form of 5- hydroxymethylcytosine. Methylation of DNA can include methylation of non-cytosine nucleotides, such as N6-methyladenine (6mA). Anomalous cfDNA methylation can be identified as hypermethylation or hypomethylation, both of which may be indicative of cancer status. As is well known in the art, DNA methylation anomalies (compared to healthy controls) can cause different effects, which may contribute to cancer.

[0147] In certain embodiments, the nucleic acid comprises a CpG site (i. e. , cytosine and guanine separated by only one phosphate group). In certain embodiments, the nucleic acid comprises a CpG island (also referred to as a “CG islands” or “CGI”) or a portion thereof, which is the target for enrichment. Because certain CGIs and certain features of certain CGIs in tumor cells tend to be different from the same CGIs or features of the CGIs in healthy cells, detection of such CGIs can be informative of a health condition. In certain embodiments, the CGI is a “cancer informative CGIs,” which is defined and described in more detail below. In certain embodiments, the CpG is an “informative CpG,” e.g., a “cancer informative CGI.” Such CGIs may have methylation patterns in tumor cells that are different from the methylation patterns in healthy cells. Accordingly, detection of a cancer informative CGI can be informative regarding a subject’s risk of developing cancer or can be indicative that the subject has cancer. Exemplary cancer informative CGIs, which can be target sequences as described herein, are identified in, e.g., Table 1 of U. S. Patent Publication 2020 / 0109456A1 and Tables 2 and 3 of WO2022 / 133315, each of which are hereby incorporated by reference in its entirety. Further exemplary cancer informative CGIs are shown in Tables 1 and 2 included herein.Attorney Docket No. HRG-027WO

[0148] In certain aspects, the nucleic acids of the invention have been treated to convert one or more unmethylated nucleotides (e.g., cytosines) to another nucleotide (a “converted nucleotide,” as used herein, such as a uracil), for example, prior to amplification. In certain embodiments, one or more unmethylated cytosines are converted to a nucleotide that pairs with adenine (e.g., the unmethylated cytosine may be converted to uracil). In certain embodiments, one or more unmethylated adenines are converted to a base that pairs with cytosine (e.g., the unmethylated adenine may be converted to inosine (I)). In certain embodiments, one or more methylated cytosines (e.g., a 5 -methylcytosine (5mC)) is converted to a thymine, which pairs with adenine. In certain embodiments, methylated cytosines are protected from conversion (e.g., deamination) during the conversion step.

[0149] After a nucleic acid has been treated to convert unmethylated, or, in some cases, methylated nucleotides, into another nucleotide, the nucleic acid may be amplified. During amplification, the converted nucleotide pairs with its complementary nucleotide, and in the next round of amplification, the complementary nucleotide pairs with a replacement nucleotide. For example, following the conversion of an unmethylated cytosine to a uracil, the nucleic acid may be amplified such that an adenine pairs with the uracil in the first round of replication, and in the second round of replication, the adenine pairs with a thymine. Accordingly, the thymine replaces the uracil in the original nucleic acid sequence, and is referred to herein as a “replacement nucleotide.” b. Bisulfite conversion

[0150] Bisulfite conversion is performed on DNA by denaturation using high heat, preferential deamination (at an acidic pH) of unmethylated cytosines, which are then converted to uracil by desulfonation (at an alkaline pH). Methylated cytosines remain unchanged on the single-stranded DNA (ssDNA) product.

[0151] In some embodiments the methods include treatment of the sample with bisulfite (e.g., sodium bisulfite, potassium bisulfite, ammonium bisulfite, magnesium bisulfite, sodium metabisulfite, potassium metabisulfite, ammonium metabisulfite, magnesium metabisulfite and the like). Unmethylated cytosine is converted to uracil through a three-step process during sodium bisulfite modification. As shown in FIG. 2A, the steps are sulphonation to convert cytosine to cytosine sulphonate, deamination to convert cytosine sulphonate to uracil sulphonateAttorney Docket No. HRG-027WO and alkali desulphonation to convert uracil sulphonate to uracil. Conversion on methylated cytosine is much slower and is not observed at significant levels in a 4-16 hour reaction. (See Clark et al. , Nucleic Acids Res. , 22(15):2990-7 (1994). ) If the cytosine is methylated it will remain a methylated cytosine. If the cytosine is unmethylated it will be converted to uracil. When the modified strand is copied, for example, through extension of a locus specific primer, a random or degenerate primer or a primer to an adaptor, a G will be incorporated in the interrogation position (opposite the C being interrogated) if the C was methylated, and an A will be incorporated in the interrogation position if the C was unmethylated and converted to U.When the double stranded extension product is amplified those Cs that were converted to Us and resulted in incorporation of A in the extended primer will be replaced by Ts during amplification. Those Cs that were not converted (z. e. , the methylated Cs) and resulted in the incorporation of G will be replaced by unmethylated Cs during amplification. c. Enzymatic conversion

[0152] In certain embodiments, the enzymatic treatment with a cytidine deaminase enzyme is used to convert cytosine to uracil. Enzymatic conversion can include an oxidation step, in which Tet methylcytosine dioxygenase 2 (TET2) catalyzes the oxidation of 5mC to 5hmC to protect methylated cytosines from conversion by subsequent exposure to a cytidine deaminase. Other protection steps known in the art can be used in addition to or in place of oxidation by TET2. After the oxidation step, the nucleic acid is treated with the cytidine deaminase to convert one or more unmethylated cytosines to uracils. As with bisulfite conversion, when the modified strand is copied, a G will be incorporated in the interrogation position (opposite the C being interrogated) if the C was methylated and an A will be incorporated in the interrogation position if the C was unmethylated. When the double stranded extension product is amplified those Cs that were converted to Us and resulted in incorporation of A in the extended primer will be replaced by Ts during amplification. Those Cs that were not modified and resulted in the incorporation of G will remain as C.

[0153] In certain embodiments the cytidine deaminase may be APOBEC. In certain embodiments the cytidine deaminase includes activation induced cytidine deaminase (AID) and apolipoprotein B mRNA editing enzymes, catalytic polypeptide-like (APOBEC). In certain embodiments, the APOBEC enzyme is selected from the human APOBEC family consisting of:Attorney Docket No. HRG-027WOAPOBEC-1 (Apol), APOBEC-2 (Apo2), AID, APOBEC-3A, -3B, -3C, -3DE, -3F, -3G, -3H and APOBEC-4 (Apo4). In certain embodiments, the APOBEC enzyme is APOBEC-seq. d. Nitrite Conversion

[0154] In certain embodiments, nitrite treatment is used to deaminate adenine and cytosine. As shown in FIG. 2B, deamination of an A results in conversion to an inosine (I), which is read by a polymerase as a G, whereas deamination of a methylated A (A6- methyladenine (6mA)) results in a nitrosylated 6mA (6mA-N0), which causes the base to be read by a polymerase as an A. Deamination of a C results in conversion to a uracil, which is read by a polymerase as a T, whereas deamination of a A^-methylcytosine (4mC) to 4mC-N0 or a 5- methylcytosine (5mC) to a T causes the base to be read by a polymerase as a C or a T, respectively. For 5mC bases, the C to T ratio at the 5mC position is about 40% higher than other cytosine positions, allowing 5mC to be differentiated from C. (See, Li et al. (2022) Genome Biology 23 : 122. )EXAMPLES

[0155] Practice of embodiments disclosed herein will be more fully understood from the foregoing examples, which are presented herein for illustrative purposes only, and should not be construed as limiting the invention in any way.Example 1 - General Methods for Enriching for Target Nucleic Acid Molecules

[0156] Generally, blocker-induced enrichment was performed by combining bisulfite- converted whole genome (WGBS) library DNA with hybridization capture reagents, targeted blockers for non-methylated sites (bisulfite converted sites) and biotinylated-probes specific for target methylated sequences. Hybridization was performed in a step-wise temperature-dependent manner in order for the targeted blockers to anneal ahead of the biotinylated-probes and then allow hybridization to continue at a set temperature for a set amount of time. Directly after hybridization incubation, streptavidin (SA) beads were combined with post-hybridization material for the biotinylated-probe-target-library-DNA to bind to the SA beads. Off-target library DNA was washed out of solution with a series of washes with increasing stringency. The captured library DNA was amplified off the SA beads using Illumina sequencing adapters. AnAttorney Docket No. HRG-027WO optional step was to identify amount of library DNA enriched using qPCR as a read-out. Finally, the nucleic acids were sequenced on an Illumina sequencer. Post-sequencing, sequenced reads were aligned to a reference to create a BAM file and subsequently the BAM file was used for all pipeline processing, including on-target enrichment percentage as well as methylation metric calling.

[0157] More specifically, the target specimen type (e. g. DNA, RNA, protein, exosomes, metabolites, etc.) was isolated from a patient’s biological source (e. g. tissue, blood, plasma, serum, saliva, feces, etc.). All target specimens were assayed for quality and quantity measurements.

[0158] The target specimens, specifically cfDNA nucleic acid molecules, underwent conversion to convert unmethylated cytosines to uracils. Bisulfite conversion was performed on DNA by denaturation using high heat, preferential deamination (at an acidic pH) of unmethylated cytosines, which were then converted to uracil by desulfonation (at an alkaline pH). Methylated cytosines remained unchanged on the single-stranded DNA (ssDNA) product.

[0159] Reference is made to FIG. 4A, which shows an example depiction of blocker- induced enrichment. Specifically, blocker-induced enrichment in PCR was performed by combining bisulfite-converted DNA (e.g., hypermethylated DNA and unmethylated DNA, as shown in FIG. 4A) with PCR reagents, targeted high Tm blockers (e.g., blockers) for nonmethylated sites (bisulfite converted sites) and primers specific for target methylated sequences. Addition of a hydrolysis probe (e. g. TaqMan) specific for targeted methylated sequences added additional specificity to target sequences. Real-time qPCR (or digital PCR) was performed for 30-50 cycles and the output was monitored for signal via fluorescence from amplified target DNA. Cycle threshold values (Ct) were recorded and exported for analysis. The delta-Ct between negative control, positive control, and sample were calculated to determine the abundance of hypermethylated DNA.

[0160] Positive results for hypermethylation target sites with the addition of blocker produced a delta-Ct between sample and negative control which was greater than or equal to 3. Negative results for hypermethylation target sites with the addition of blocker produced a result of delta-Ct between sample and negative control which was less than 2. Inconclusive results for hypermethylation target sites with the addition of a blocker produced a result of delta-Ct between the sample and negative control which was greater than or equal to 2 but less than 3. FIGs. 4B-Attorney Docket No. HRG-027WO4D show example qPCR graphs showing theoretical relative detected fluorescence across amplification cycles for methylated and unmethylated DNA. Specifically, FIG. 4B shows that the change in cycle threshold (Ct) for methylated DNA does not change in the presence or absence of blockers. FIG. 4C shows a shift in the Ct value for unmethylated DNA in the presence of blockers. Here, amplification of the unmethylated DNA was delayed. FIG. 4D depicts a scenario in which unmethylated DNA was not amplified or not detected by hydrolysis probe, thereby resulting in a significant change in Ct value. Here, this scenario arises in the presence of blockers with a polymerase termination modification.Experimental ProtocolsOligo Resuspension1. Disinfect the PCR hood in the pre-PCR lab with DNA Away for 10 minutes, followed by 70% ethanol. Optionally, run the UV light for 30 minutes prior to disinfection.2. Spin down the lyophilized oligonucleotides (e.g., blockers with a polymerase terminating modification).3. Perform vortex, centrifugation, and incubation.4. Prepare the desired working dilution for the forward primer and reverse primer mixture, such as 10 pM. For 10 pM forward and reverse primer mixture, add 10 pL of each primer to 80 pL TLE.5. Prepare the desired working dilution for the TaqMan probe, such as 10 pM. For 10 pM probe, add 10 pL of probe to 90 pL TLE.6. Store oligo stocks at -20 °C. Bring the oligo working dilutions to the post-PCR lab and store at -20 °C.Prepare qPCR Plate1. Prepare a qPCR plate layout which depicts the well location for each qPCR condition within a 384- well plate. The table 3 below shows an example of an annotated 384- well qPCR plate layout. Each well provides sufficient information to distinguish the qPCR components used in each well. In this example, three blocker sequences are tested across two qPCR assays at two gBlock DNA template levels.Table 3. Example of an annotated 384- well qPCR plate layout. Each well provides sufficient information to distinguish the qPCR components used in each well. In this example, five blocker types are tested across two qPCR assays at two gBlock DNA template levels.Attorney Docket No. HRG-027WOLanes 1 and 3: No blocker control. Lanes 5 and 7, CpG4(+): Blocker #1. Lanes 5 and 7, CpG4(- ): Blocker #5. Lanes 9 and 11, CpG4(+): Blocker #3. Lanes 9 and 11, CpG4(-): Blocker #7.1. Prepare the qPCR mixtures (N = 3 per condition). a. A standard qPCR mixture using IDT’s PrimeTime Gene Expression Master Mix and a single-plex qPCR with a single TaqMan probe, in 10 pL and 20 pL final volume, is shown in the Table 4 below. b. A standard qPCR mixture using Bio-Rad’s SYBR Green Master Mix and a single- plex qPCR, in 10 pL and 20 pL final volume, is shown in Table 5 below. c. Multiple N = 1 by the total number of wells to create a bulk mixture. The volumes will change if using different reagent concentrations or adding more reagents, such as in a multiplex reaction. Use Cl V1=C2V2 when modifying the volume required for a particular reagent concentration.Table 4. Standard single-plex qPCR mixture using IDT’s Prime Time Gene Expression Master Mix.Attorney Docket No. HRG-027WOTable 5. Standard single-plex qPCR mixture using Bio-Rad’s SYBR Green Supermix.2. Add 10 p L of each qPCR mixture to the qPCR plate based on the qPCR plate layout. a. Briefly vortex and centrifuge the N = 3 qPCR mixtures prior to loading on the plate.3. Tightly seal the qPCR plate with an optical seal, especially around the perimeter of the plate and across the top of the plate.Run the qPCR Plate1. Place the qPCR plate in the CFX 384 or CPF Opus 384 instrument in the correct orientation.2. Run the standard qPCR protocol as follows: 95 °C for 3 minutes, 45 cycles of 95 °C for 30 seconds, 60 °C for 45 seconds with a plate reader turned ON.Example 2 -Blockers with a Polymerase Terminating Modification EnableDifferential Enrichment of Nucleic Acid Molecules

[0161] Generally, the experimental protocols described in Example 1 were performed to evaluate the use of blockers with a polymerase terminating modification (PTB) for differentialAttorney Docket No. HRG-027WO enrichment of nucleic acid molecules. CpG-dense regions that are cancer-informative were identified. These CpG-dense regions exhibited increased levels of hypermethylation in cancer samples and hypomethylation in non-cancer samples. Quantitative bisulfite-specific (qBSP) or methylation-specific qPCR (qMSP) assays were designed with hydrolysis probes covering these target CpG sites. Blockers with a polymerase terminating modification were designed to specifically bind to the unmethylated species, and outcompete the forward primer or reverse primer through a high Tm differential, thereby displacing the primer and preventing subsequent amplification. Due to the polymerase terminating 3’ end modification, the PTB blocker will not elongate and the unmethylated fragment is not amplified. qBSP or qMSP assays were run both with and without polymerase terminating modification blockers and the percentage of methylated DNA was estimated by comparing the amount of methylated copies to total copies.

[0162] Six blockers were developed and tested. The blockers each included polymerase terminating modifications as shown in the following Table 6. Underlined nucleotides indicate nucleotides that overlap with a primer sequence. The Tm difference of the blocker to the primer it overlaps in sequence with, as well as other characteristics, are summarized in Table 6. Furthermore, Table 7a below shows the relative location of each blocker sequence relative to 1) CpG sites, 2) probe (e.g., Taqman probe) binding location, 3) and forward / reverse primer binding locations. Specifically, the blocker sequence in the “Relative Sequences” column of Table 7a is shown in parentheticals. For example, for Blocker 8, the blocker sequence is denoted between brackets, “[ X].” Corresponding CpG sites (e.g., CpG sites on the target sequence) are shown in bold. For example, for Blocker 8, there are a total of 3 CpG sites in the relative target sequence, each bolded as “TG” (which is derived from a corresponding unmethylated CpG site as the unmethylated “C” was converted to a uracil and the complementary base is thymine “T”). For Blocker 8, the full probe binding sequence is “XX, ” where the final two thymine nucleotides overlap with the first two nucleotides of the blocker sequence. Forward and reverse primer binding sequences are shown in underline.Attorney Docket No. HRG-027WOTable 6: Example blocker sequences with polymerase terminating modifications.C3 indicates a C3 spacer and is not a nucleotide that is part of the sequence; ddC indicates the modified nucleotide di deoxy C-3’; underlined nucleotides indicate nucleotides that overlap with a primer sequence. Bold “T” indicates a position corresponding to an unmethylated cytosine that was converted to a uracil by bisulfite conversion in the target sequence.Table 7a: Example blocker sequence relative to CpG sites and forward / reverse primer binding locations.Attorney Docket No. HRG-027WOTable 7b: Exemplary primer sequencesUnderline nucleotides in Long Primer sequence are not included in the Short Primer sequence.Attorney Docket No. HRG-027WOBlockers Bind Differently to Unmethylated Candidate Sequences

[0163] Blocker #8, #9, and #10 with a C3 spacer were tested with unmethylated CpG4 (-) gBlock. gBlocks are double-stranded DNA fragments. The electrophoresis gel results are shown in Figure 5. Blocker #10 showed high blocking efficiency, and had an additional three base overlap and an additional 3 °C Tm difference with the forward primer compared to Blocker #9, indicating the amount of overlap with the primer and / or the delta Tm influenced PTB blocking efficiency. Blocker #8 and Blocker #9, which had ATm = 4 °C, blocked the amplification of unmethylated DNA to a lesser extent in comparison to Blocker #10.

[0164] For each blocker, two 3’ end modifications which terminate polymerase extension were evaluated: a C3 Spacer and a ddC. The addition of a ddC to the 3’ end of the oligo, as described in Table 6, was supposed to increase the Tm by about 0.5 to 2.2 °C. This potential Tm increase from ddC did not result in effective blocking using Blocker #9, even though the delta Tm was > 5 °C. It is likely that the addition of ddC does not result in a “true” Tm increase as the ddC is mismatched with the template DNA. Thus, if using 3’ end ddC in the future, the delta Tm requirement should be met without the consideration of the ddC in the Tm calculation. If ddC is a match to the template DNA (e.g., the template DNA contains a guanine in this position), the influence of ddC on delta Tm should be evaluated.

[0165] Blocker #10, with a C3 spacer, was then tested with various assay Tms to determine the percent inhibition at each tested temperature. Parallel reactions were run with no blocker for comparison. The results are shown in FIG. 6A and FIG. 6B and summarized in Table 8 below. Blocker #10 showed the highest inhibition of amplification of unmethylated DNA at 52°C.

[0166] % Inhibition was calculated with the following formula:Table 8.Attorney Docket No. HRG-027WO

[0167] Blocker #10 was then tested with the original full length BSP primers (ATm = 3 °C) and the shortened BSP primers (ATm = 7. 1 °C), the sequences of which are summarized in Table 7b, which were designed to maximize ATm by lowering the overlapping primer Tm. The BSP primers were shortened at the 5’ end, so the 10-base overlap between Blocker #10 and the forward primer was maintained. The electrophoresis gel results are shown in FIG. 7. Blocker #10 did not block DNA amplification as efficiently with the full length BSP primers compared to the shortened primers, indicating that the high Tm differential between the blocker and forward primer influences optimal blocking efficiency.

[0168] Next, the annealing temperature and blocker-to-primer ratio were tested for Blocker #10 (primer concentration = 500 nM; blocker concentration = 2. 5 uM (5X), 7. 5 uM (15X), or 25 uM (50X)) using unmethylated, converted gBlock DNA. The electrophoresis gel results are shown in FIG. 8. The highest blocking efficiency across all blocker concentrations tested, based on the target amplicon product molarity (region between 75 bases and 150 bases), was at 56 °C. However, the BSP primers showed specificity issues with gDNA at this lower annealing temperature; thus, the optimal annealing temperature must be determined for both the primer set and blocker set used. As the blocker concentration increased relative to the primer concentration, the blocking efficiency increased.

[0169] SYBR Green qPCR was then utilized as an additional readout of the blocking ability of Blocker 10. Melt Curve Analysis and electrophoresis gels were performed to confirm primer dimers were not contributing to SYBR Green fluorescence.

[0170] The results of the SYBR Green qPCR are summarized in Table 9 below. Blocker #10 delayed the unmethylated DNA Ct by about 12 cycles at > 5X concentration of blocker to primer. Blocker #10 maintained specificity for unmethylated DNA, without the inhibition of methylated DNA, until the blocker-to-primer ratio was between 15X and 50X. Both C3 and ddC 3’ end modification blocked with similar efficiency and specificity at concentrations < 50X concentration of blocker to primer. Higher blocking observed with ddC than C3 at 50X. BoldAttorney Docket No. HRG-027WO values indicate observed blocking of the amplification of methylated DNA. Italicized values indicate observed blocking of the amplification of unmethylated DNA.Table 9.

[0171] Further, MSP and BSP assays were run with a TaqMan probe. The TaqMan probe’s binding region was similar to Blocker #10’ s binding region. Reactions were run at the various Tm as run for testing the percent inhibition of Blocker #10 and Ct cycles were measured. The results are summarized in Table 10 and FIG. 9. The BSP probe and Blocker #10 overlap on the same binding site. It was observed the BSP probe outcompeted the binding of Blocker #10 at some of the tested annealing temperatures, resulting in a difference of Ct cycles of less than 2. The MSP probe bound to the opposite strand that Blocker #10 does, so the binding sites do not overlap, however, the MSP probe is the reverse compliment of the blocker, which caused dimerization and impacted qPCR fluorescence, as can be seen by a Ct difference of 2 or below for various annealing temperatures. Therefore, TaqMan probes should be designed to notAttorney Docket No. HRG-027WO significantly overlap to the binding site, or complementary of the binding site, of the blocker with a polymerase terminating modification.Table 10.

[0172] In summary, the results show that a higher Tm differential between the PTB blocker and the overlapping correlated with blocking efficiency. A blocker Tm > 5 °C higher than the overlapping primer Tm worked well. Increasing blocker concentration also improved blocking efficiency. At a certain blocker-to-primer concentration, between 15X and 50X, nonspecific blocking occurred. At and below 15X blocker-to-primer concentration, the PTB blocker did not inhibit methylated DNA amplification. Lowering the PCR annealing temperature improved blocking efficiency. The annealing temperature influences the blocker efficiency and PCR efficiency, and should be optimized for both.Attorney Docket No. HRG-027WOTablesTable 1. Genomic ranges of low background regions including a plurality of CpG sites mapped to human genome, hgl9. The “Genomic Coordinate Start” and “Genomic Coordinate End” columns indicate the beginning and end, respectively of a range of genomic locations within a chromosome (“Chrom. ”).Attorney Docket No. HRG-027WOAttorney Docket No. HRG-027WOTable 2: Exemplary Target RegionsHuman universal genomic locations and CpG sites mapped to human genome, hg38. The “Start” and “End” columns indicate the beginning and end, respectively of a range of a target region within a chromosome.Attorney Docket No. HRG-027WOAttorney Docket No. HRG-027WOAttorney Docket No. HRG-027WOAttorney Docket No. HRG-027WOAttorney Docket No. HRG-027WOAttorney Docket No. HRG-027WOAttorney Docket No. HRG-027WOAttorney Docket No. HRG-027WOAttorney Docket No. HRG-027WOAttorney Docket No. HRG-027WOAttorney Docket No. HRG-027WOAttorney Docket No. HRG-027WOAttorney Docket No. HRG-027WOAttorney Docket No. HRG-027WOAttorney Docket No. HRG-027WOAttorney Docket No. HRG-027WOAttorney Docket No. HRG-027WOAttorney Docket No. HRG-027WOAttorney Docket No. HRG-027WOAttorney Docket No. HRG-027WOAttorney Docket No. HRG-027WOAttorney Docket No. HRG-027WOAttorney Docket No. HRG-027WOAttorney Docket No. HRG-027WOAttorney Docket No. HRG-027WOAttorney Docket No. HRG-027WOAttorney Docket No. HRG-027WOAttorney Docket No. HRG-027WOAttorney Docket No. HRG-027WOAttorney Docket No. HRG-027WO

Claims

Attorney Docket No. HRG-027WOCLAIMSWHAT IS CLAIMED IS:

1. A method for performing differential enrichment of nucleic acid molecules, the method comprising: a) providing a mixture of nucleic acid molecules comprising: a first set of nucleic acid molecules comprising a target sequence derived from a sequence comprising one or more methylated CpG sites; and a second set of nucleic acid molecules comprising a candidate sequence derived from a sequence comprising one or more non-methylated CpG sites, b) providing a blocker that binds to the candidate sequence, or a portion thereof, or the complementary sequence thereof, of the second set of nucleic acid molecules, the blocker comprising a polymerase terminating modification; c) selectively enrich for the first set of nucleic acid molecules comprising the target sequence in comparison to the second set of nucleic acid molecules comprising the candidate sequence, wherein the blocker comprising the polymerase terminating modification prevents or reduces enrichment of the second set of nucleic acid molecules comprising the candidate sequence; and d) detecting the selectively enriched first set of nucleic acid molecules.

2. The method of claim 1, wherein the polymerase terminating modification is selected from the group consisting of 3’ ddC, 3’ ddG, 3’ ddA, 3’ ddT, 3’ inverted dT, 3’ C3 spacer, 3’ amino, and 3’ phosphorylation.

3. The method of claim 1 or 2, wherein the polymerase terminating modification is 3’ ddC.

4. The method of claim 1 or 2, wherein the polymerase terminating modification is 3’ C3.

5. The method of claim 1 or 2, wherein the candidate sequence comprises at least two unmethylated CpG sites or the complementary sequence thereof.

6. The method of claim 1 or 2, wherein the candidate sequence comprises two unmethylated CpG sites or the complementary sequence thereof.

7. The method of claim 1 or 2, wherein candidate sequence comprises three unmethylated CpG sites or the complementary sequence thereof.Attorney Docket No. HRG-027WO8. The method of any one of claims 1-7, wherein selectively enriching the first set of nucleic acid molecules comprises performing one or more of PCR, RT-PCR, digital PCR, qPCR, nicking endonuclease amplification (NEAR), transcription-mediated amplification (TMA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HAD), strand displacement amplification (SDA), sequencing library PCR, hybrid capture, microarrays, next generation sequencing (NGS), or hybrid capture PCR.

9. The method of any one of claims 1-8, wherein selectively enriching for the first set of nucleic acid molecules comprises providing a forward primer and a reverse primer for performing nucleic acid amplification.

10. The method of claim 9, wherein when the forward primer or the reverse primer is bound to a sequence of the second set of nucleic acid molecules, an end of the forward primer or the reverse primer overlaps with the sequence bound by the blocker by at least 3 nucleotides, at least 5 nucleotides, at least 7 nucleotides, at least 9 nucleotides, or at least 11 nucleotides.

11. The method of claim 9 or 10, wherein when the forward primer or the reverse primer is bound to a sequence of the second set of nucleic acid molecules, an end of the forward primer or the reverse primer overlaps with the sequence bound by the blocker by 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, or 11 nucleotides.

12. The method of any one of claims 9-11, wherein when the forward primer or the reverse primer is bound to a sequence of the second set of nucleic acid molecules, an end of the forward primer or the reverse primer overlaps with the sequence bound by the blocker by 7 nucleotides.

13. The method of any one of claims 9-11, wherein when the forward primer or the reverse primer is bound to a sequence of the second set of nucleic acid molecules, an end of the forward primer or the reverse primer overlaps with the sequence bound by the blocker by 10 nucleotides.

14. The method of any one of claims 1-11, wherein the blocker comprises a sequence that shares at least 85% identity with any one of SEQ ID NOs: 1-6.

15. The method of any one of claims 1-11 or 14, wherein the blocker comprises a sequence that shares at least 95% identity with any one of SEQ ID NOs: 1-6.Attorney Docket No. HRG-027WO16. The method of any one of claims 9-15, wherein the blocker, when bound to the candidate sequence, has a melting temperature at least 5°C higher, at least 6°C higher, at least 7°C, or at least 8°C higher than the melting temperature of the forward primer or the reverse primer.

17. The method of any one of claims 9-15, wherein the blocker, when bound to the candidate sequence, has a melting temperature at least 6°C higher than the melting temperature of the forward primer or the reverse primer.

18. The method of any one of claims 9-15, wherein the blocker, when bound to the candidate sequence, has a melting temperature at least 7°C higher than the melting temperature of the forward primer or the reverse primer.

19. The method of any one of claims 9-15, wherein the blocker, when bound to the candidate sequence, has a melting temperature at least 8°C higher than the melting temperature of the forward primer or the reverse primer.

20. The method of any one of claims 1-17, wherein the blocker comprises between 20 and 35 nucleosides, optionally between 23-33 nucleosides.

21. The method of any one of claims 1-20, further comprising: between step (b) and step (c), exposing the blocker and the candidate sequence to one or more temperatures, thereby enabling the blocker to bind to the candidate sequence or complementary sequence thereof.

22. The method of claim 21, wherein the one or more temperatures comprises a temperature between 50°C and 65°C, optionally about 56°C.

23. The method of any one of claims 1-20, wherein the first set of nucleic acid molecules and / or the second set of nucleic acid molecules are DNA.

24. The method of claim 23, wherein the DNA is cell-free DNA.

25. The method of any one of claims 1 -20, wherein the first set of nucleic acid molecules and / or the second set of nucleic acid molecules are RNA.

26. The method of any one of claims 1 -24, wherein the first set of nucleic acid molecules and / or the second set of nucleic acid molecules have been treated using bisulfite conversion to convert unmethylated cytosines to uracil.

27. The method of any one of claims 1 -24, wherein the first set of nucleic acid molecules and / or the second set of nucleic acid molecules have been treated using enzymatic conversion to convert unmethylated cytosines to uracil.Attorney Docket No. HRG-027WO28. The method of claim 27, wherein the enzymatic conversion is selected from TET2 oxidation of cytosines and APOBEC conversion of cytosines.

29. The method of any one of claims 1 -28, wherein the first set of nucleic acid molecules and / or the second set of nucleic acid molecules were obtained from a sample.

30. The method of claim 29, wherein the sample comprises a tissue sample, a blood sample, a stool sample, a urine sample, a mucous sample, or a saliva sample.

31. The method of any one of claims 1-30, wherein the target sequence comprises at least a CpG island, or a portion thereof.

32. The method of any one of claims 1-31, further comprises: either simultaneous with step (b) or after step (b), providing a second blocker that binds to a different candidate sequence, or a portion thereof, of the second set of nucleic acid molecules, the second blocker comprising a polymerase terminating modification.

33. The method of any one of claims 1-31, further comprises: either simultaneous with step (b) or after step (b), providing a probe capable of hybridizing to one or both of the target sequence, or a portion thereof, of the first set of nucleic acid molecules and the candidate sequence, or a portion thereof, of the second set of nucleic acid molecules.

34. The method of claim 33, further comprising hybridizing the probe to the target sequence, or a portion thereof of the first set of nucleic acid molecules, wherein selectively enriching for the first set of nucleic acid molecules comprises enriching for the first set of nucleic acid molecules using the probe hybridized to the target sequence, or a portion thereof, of the first set of nucleic acid molecules.

35. The method of claim 34, wherein enriching for the first set of nucleic acid molecules using the probe comprises performing hybrid capture.

36. A method for performing differential enrichment of nucleic acid molecules, the method comprising: a) providing nucleic acid molecules comprising a target sequence derived from a sequence comprising one or more methylated CpG sites; b) providing a blocker capable of binding to a candidate sequence, or a portion thereof, or the complementary sequence thereof, the candidate sequence only differing from the targetAttorney Docket No. HRG-027WO sequence at nucleotides corresponding to the one or more methylated CpG sites, wherein the blocker comprises a polymerase terminating modification; c) enriching for the nucleic acid molecules comprising the target sequence, wherein the blocker comprising a polymerase terminating modification does not prevent enrichment of the nucleic acid molecules comprising the target sequence; and d) detecting the enriched nucleic acid molecules.

37. The method of claim 36, wherein the polymerase terminating modification selected from the group consisting of 3’ ddC, 3’ ddG, 3’ ddA, 3’ ddT, 3’ inverted dT, 3’ C3 spacer, 3’ amino, and 3’ phosphorylation.

38. The method of claim 36 or 37, wherein the polymerase terminating modification is 3’ ddC.

39. The method of claim 36 or 37, wherein the polymerase terminating modification is 3’ C3.

40. The method of claim 36 or 37, wherein the candidate sequence comprises least two unmethylated CpG sites or the complementary sequence thereof.

41. The method of claim 36 or 37, wherein the candidate sequence comprises two unmethylated CpG sites or the complementary sequence thereof.

42. The method of claim 36 or 37, wherein the candidate sequence comprises three unmethylated CpG sites or the complementary sequence thereof.

43. The method of any one of claims 36-42, wherein enriching for the nucleic acid molecules comprises performing one or more of one or more of PCR, RT-PCR, digital PCR, nicking endonuclease amplification (NEAR), transcription-mediated amplification (TMA), loop- mediated isothermal amplification (LAMP), helicase-dependent amplification (HAD), strand displacement amplification (SDA), sequencing library PCR, hybrid capture, or hybrid capture PCR.

44. The method of any one of claims 36-43, wherein enriching for the nucleic acid molecules comprises providing a forward primer and a reverse primer for performing nucleic acid amplification.

45. The method of claim 44, wherein when the forward primer or the reverse primer is bound to a sequence of the second set of nucleic acid molecules, an end of the forward primer or the reverse primer overlaps with the sequence bound by the blocker by at least 3 nucleotides, at least 5 nucleotides, at least 7 nucleotides, at least 9 nucleotides, or at least 11 nucleotides.Attorney Docket No. HRG-027WO46. The method of any one of claims 44-45, wherein when the forward primer or the reverse primer is bound to a sequence of the second set of nucleic acid molecules, an end of the forward primer or the reverse primer overlaps with the sequence bound by the blocker by 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, or 11 nucleotides.

47. The method of any one of claims 44-46, wherein when the forward primer or the reverse primer is bound to a sequence of the second set of nucleic acid molecules, an end of the forward primer or the reverse primer overlaps with the sequence bound by the blocker by 7 nucleotides.

48. The method of any one of claims 44-46, wherein when the forward primer or the reverse primer is bound to a sequence of the second set of nucleic acid molecules, an end of the forward primer or the reverse primer overlaps with the sequence bound by the blocker by 10 nucleotides.

49. The method of claim 36 or 37, wherein the blocker comprises a sequence that shares at least 85% identity with any one of SEQ ID NOs: 1-6.

50. The method of claim 36 or 37, wherein the blocker consists of a sequence of any one of SEQ ID NOs: 1-6.

51. The method of any one of claims 36-50, wherein the blocker comprises between 20 and 35 nucleosides, optionally between 23-33 nucleosides.

52. The method of any one of claims 36-51, further comprising: between step (b) and step (c), exposing the blocker to one or more temperatures.

53. The method of claim 52, wherein the one or more temperatures comprises a temperature between 50°C and 65°C, optionally about 56°C.

54. The method of any one of claims 36-53, wherein the nucleic acid molecules are DNA.

55. The method of claim 54, wherein the DNA is cell-free DNA.

56. The method of any one of claims 36-55, wherein the nucleic acid molecules are RNA.

57. The method of any one of claims 36-56, wherein the nucleic acid molecules have been treated using bisulfite conversion to convert unmethylated cytosines to uracil.

58. The method of any one of claims 36-56, wherein the nucleic acid molecules have been treated using enzymatic conversion to convert unmethylated cytosines to uracil.Attorney Docket No. HRG-027WO59. The method of claim 58, wherein the enzymatic conversion is selected from TET2 oxidation of cytosines and APOBEC conversion of cytosines.

60. The method of any one of claims 36-59, wherein the nucleic acid molecules were obtained from a sample.

61. The method of claim 60, wherein the sample comprises a tissue sample, a blood sample, a stool sample, a urine sample, a mucous sample, or a saliva sample.

62. The method of any one of claims 36-59, wherein the target sequence comprises at least a CpG island, or a portion thereof.

63. The method of any one of claims 36-62, further comprises: either simultaneous with step (b) or after step (b), providing a probe capable of hybridizing to the target sequence, or a portion thereof, of the nucleic acid molecules.

64. The method of claim 63, further comprising hybridizing the probe to the target sequence, or a portion thereof, or the complementary sequence thereof, of the nucleic acid molecules, wherein selectively enriching for the first set of nucleic acid molecules comprises enriching for the nucleic acid molecules using the probe hybridized to the target sequence, or a portion thereof, of the nucleic acid molecules.

65. The method of claim 64, wherein enriching for the nucleic acid molecules using the probe comprises performing hybrid capture.

66. A method for performing differential enrichment of nucleic acid molecules, the method comprising: a) providing nucleic acid molecules comprising a candidate sequence derived from a sequence comprising one or more non-methylated CpG sites; b) providing a blocker capable of binding to the candidate sequence, or a portion thereof, or the complementary sequence thereof, wherein the blocker comprises a polymerase terminating modification; and c) exposing the nucleic acid molecules comprising the candidate sequence to conditions suitable for nucleic acid enrichment, wherein the blocker comprising a polymerase terminating modification binds to the candidate sequence, or a portion thereof, or the complementary sequence thereof, and prevents or reduces enrichment of nucleic acid molecules comprising the candidate sequence.Attorney Docket No. HRG-027WO67. The method of claim 66, wherein the polymerase terminating modification selected from the group consisting of 3 ’ddC, 3’ inverted dT, 3’ C3 spacer, 3’ amino, and 3’ phosphorylation.

68. The method of claim 66 or 67, wherein the polymerase terminating modification is 3’ ddC.

69. The method of claim 66 or 67, wherein the polymerase terminating modification is 3’ C3.

70. The method of claim 66 or 67, wherein the blocker sequence binds to at least two unmethylated CpG sites or the complementary sequence thereof.

71. The method of claim 66 or 67, wherein the candidate sequence comprises two unmethylated CpG sites or the complementary sequence thereof.

72. The method of claim 66 or 67, wherein the candidate sequence comprises three unmethylated CpG sites or the complementary sequence thereof.

73. The method of any one of claims 66-72, wherein enriching for the nucleic acid molecules comprises performing one or more of one or more of PCR, RT-PCR, digital PCR, nicking endonuclease amplification (NEAR), transcription-mediated amplification (TMA), loop- mediated isothermal amplification (LAMP), helicase-dependent amplification (HAD), strand displacement amplification (SDA), sequencing library PCR, hybrid capture, or hybrid capture PCR.

74. The method of claim 73, wherein selectively enriching for the first set of nucleic acid molecules comprises providing a forward primer and a reverse primer for performing nucleic acid amplification.

75. The method of claim 74, wherein when the forward primer or the reverse primer is bound to a sequence of the second set of nucleic acid molecules, an end of the forward primer or the reverse primer overlaps with the sequence bound by the blocker by at least 3 nucleotides, at least 5 nucleotides, at least 7 nucleotides, at least 9 nucleotides, or at least 11 nucleotides.

76. The method of claim 74 or 75, wherein when the forward primer or the reverse primer is bound to a sequence of the second set of nucleic acid molecules, an end of the forward primer or the reverse primer overlaps with the sequence bound by the blocker by 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, or 11 nucleotides.Attorney Docket No. HRG-027WO77. The method of any one of claims 74-76, wherein when the forward primer or the reverse primer is bound to a sequence of the second set of nucleic acid molecules, an end of the forward primer or the reverse primer overlaps with the sequence bound by the blocker by 7 nucleotides.

78. The method of any one of claims 74-77, wherein when the forward primer or the reverse primer is bound to a sequence of the second set of nucleic acid molecules, an end of the forward primer or the reverse primer overlaps with the sequence bound by the blocker by 10 nucleotides.

79. The method of any one of claims 66 -78, wherein the blocker comprises a sequence that shares at least 85% identity with any one of SEQ ID NOs: 1-6.

80. The method of any one of claims 66 -78, wherein the blocker comprises a sequence that shares at least 90% identity with any one of SEQ ID NOs: 1-6.

81. The method of any one of claims 66 -78, wherein the blocker comprises a sequence of any one of SEQ ID NOs: 1-6.

82. The method of any one of claims 66-81, wherein the blocker comprises between 20 and 35 nucleosides, optionally between 23-33 nucleosides.

83. The method of any one of claims 66-82, further comprising: between step (b) and step (c), exposing the blocker to one or more temperatures, thereby enabling the blocker to bind to the candidate sequence or the complementary sequence thereof.

84. The method of claim 83, wherein the one or more temperatures comprises a temperature between 50°C and 65°C, optionally about 56°C.

85. The method of any one of claims 66-84, wherein the nucleic acid molecules are DNA.

86. The method of claim 85, wherein the DNA is cell-free DNA.

87. The method of any one of claims 66-84, wherein the nucleic acid molecules are RNA.

88. The method of any one of claims 66-87, wherein the nucleic acid molecules have been treated using bisulfite conversion to convert unmethylated cytosines to uracil.

89. The method of any one of claims 66-87, wherein the nucleic acid molecules have been treated using enzymatic conversion to convert unmethylated cytosines to uracil.

90. The method of claim 89, wherein the enzymatic conversion is selected from TET2 oxidation of cytosines and APOBEC conversion of cytosines.Attorney Docket No. HRG-027WO91. The method of any one of claims 66-90, wherein the nucleic acid molecules were obtained from a sample.

92. The method of claim 91, wherein the sample comprises a tissue sample, a blood sample, a stool sample, a urine sample, a mucous sample, or a saliva sample.

93. The method of any one of claims 66-92, wherein the target sequence comprises at least a CpG island, or a portion thereof.

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