Methylation detection array and kit

Abstract

A methylation detection array includes a substrate with depressions, a bead in each depression, and a sample probe attached to each bead. The sample probes include probe sets (i.e., unmethylated probe and corresponding methylated probe). Unmethylated and methylated probes are each based on: i) a bisulfite or enzymatically converted top strand sequence of a corresponding top strand sequence that contains a high minor allele frequency single nucleotide polymorphism (SNP) where a reference is [G] followed by [CG]; or ii) a bisulfite or enzymatically converted bottom strand sequence of a corresponding bottom strand sequence that contains a high minor allele frequency SNP where a reference is [G] followed by [N] and preceded by [CG]; or iii) a bisulfite or enzymatically converted bottom strand sequence of a corresponding bottom strand sequence that contains a high minor allele frequency SNP where a reference is [C] followed by [G] and preceded by [CG].

Description

METHYLATION DETECTION ARRAY AND KITCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Serial Number 63 / 579,467, filed August 29, 2023, the content of which is incorporated by reference herein in its entirety.REFERENCE TO SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on August 21 , 2024, is named ILI266BPCTJP- 2651 -PCT_Sequence_Listing. xml and is 27,410,948 bytes in size.BACKGROUND

[0003] Deoxyribonucleic acid (DNA) methylation is an epigenetic mechanism in the mammalian genome that involves the transfer of a methyl group or a hydroxymethyl onto the C5 position of the cytosine to form, respectively, 5- methylcytosine or 5-hydroxymethylcytosine. DNA methylation regulates gene expression by recruiting proteins involved in gene repression or by inhibiting the binding of transcription factor(s) to DNA. DNA methylation affects the regulation of gene expression in development, in differentiation, and in diseases, such as multiple sclerosis, diabetes, schizophrenia, and cancers.SUMMARY

[0004] The methylation array disclosed herein includes at least some sample probes that are based on respective portions of the human genome containing high minor allele frequency single nucleotide polymorphisms (SNPs) in one of two patterns. The probes enable background normalization, correct methylation calls, and correct genotyping.BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Features of examples of the present disclosure will become apparent by reference to the following detailed description and drawings, in which like reference numerals correspond to similar, though perhaps not identical, components. For the sake of brevity, reference numerals or features having a previously described function may or may not be described in connection with other drawings in which they appear.

[0006] Fig. 1 A depicts a perspective view of an example of the methylation array disclosed herein;

[0007] Fig. 1 B depicts a cross-sectional view taken along line 1 B-1 B of Fig. 1 A;

[0008] Fig. 2 depicts an example of a double stranded DNA where the top strand includes one example of the high minor allele frequency single nucleotide polymorphism where a reference is [G] followed by [CG], and the methylated and unmethylated bisulfite converted strands that can be formed from the top strand;

[0009] Fig. 3 depicts another example of a double stranded DNA where the bottom strand includes one example of the high minor allele frequency single nucleotide polymorphism where a reference is [C] followed by [G] and preceded by [CG], and the methylated and unmethylated bisulfite converted strands that can be formed from the bottom strand;

[0010] Fig. 4 depicts another example of a double stranded DNA where the bottom strand includes one example of the high minor allele frequency single nucleotide polymorphism where a reference is [G] followed by [G] and preceded by [CG], and the methylated and unmethylated bisulfite converted strands that can be formed from the bottom strand;

[0011] Fig. 5 depicts an example of two example target strands, the strands after bisulfite conversion, and the strands after amplification, and probes generated for the strands; and

[0012] Fig. 6. depicts an example of a target strand and the strand after enzymatic conversion.DETAILED DESCRIPTION

[0013] In some methylation assays, methylated cytosines can be distinguished from non-methylated cytosines based on their differential reactivity with bisulfite, in which case the latter are converted to uracil (U) and the former are protected from conversion. In other methylation assays, methylated cytosines can be distinguished from non-methylated cytosines based on their differential reactivity with an altered cytidine deaminase, in which case the former are converted to thymine (T) by deamination at a greater rate than conversion of cytosine (C) to uracil (U) by deamination. In the examples set forth herein, nucleic acids in a sample are treated with bisulfite or enzymatic deamination, and are detected using an example of the methylation array disclosed herein. With the example methylation arrays, the methylation of genomic CpG positions in a sample can be detected using an array of sample probes. It is to be understood that a genomic CpG position refers to a locus where a cytosine nucleotide (C) is followed by a guanine nucleotide (G) in the 5’ to 3’ direction, and where the C and G are linked by a phosphate group.

[0014] In addition to methylation detection, the sample probes in the methylation arrays disclosed herein can also be used for background normalization and for genotyping the nucleotide adjacent to the [CG] of the probe, which has been selected for its known high minor allele frequency single nucleotide polymorphisms.

[0015] An example of the methylation detection array 10 is depicted in Fig. 1A. The methylation detection array 10 includes a substrate 12 having a plurality of depressions 14 defined therein; a plurality of beads 16, each of the plurality of beads 16 positioned within one of the plurality of depressions 14; and a plurality of sample probes (e.g., probes 18 and 20) respectively attached to the plurality of beads 16, the plurality of sample probes including a plurality of probe sets, wherein each of the plurality of probe sets includes an unmethylated probe 18 and a corresponding methylated probe 20; and the unmethylated probe 18 and the corresponding methylated probe 20 are: i) each based on a bisulfite converted or enzymatically converted top strand sequence of a corresponding top strand sequence that contains a high minor allele frequency single nucleotide polymorphism where a reference is [G] followed by [CG]; or ii) each based on a bisulfite converted or enzymatically convertedbottom strand sequence of a corresponding bottom strand sequence that contains a high minor allele frequency single nucleotide polymorphism where a reference is [G] followed by [N] and preceded by [CG] (where N is A, T, C, or G); or iii) each based on a bisulfite converted or enzymatically converted bottom strand sequence of a corresponding bottom strand sequence that contains a high minor allele frequency single nucleotide polymorphism where a reference is [C] followed by [G] and preceded by [CG],

[0016] It is to be understood that the terms “precede” and variations thereof and “follow” and variations thereof are used herein in reference to the positioning of nucleotide(s) in a nucleotide sequence that is in the 5’— >3’ direction. As an example, a [C] followed by a [G] is 5’— » [CG] — >3’ and a [C] preceded by a [CG] is 5’— »■ [CGC] — >3’.

[0017] Additionally the phrase “high minor allele frequency” refers to a minor allele frequency that is greater than 0.01 in either the TOPMed program (Trans-Omics for Precision Medicine) or the 1000 Genomes Project.

[0018] The substrate 12 may be a single layer base support or a multi-layered structure. In either instance, the substrate 12 includes depressions 14 defined at the surface.

[0019] When the substrate 12 is a single layer base support, examples of suitable materials for the substrate 12 include siloxanes (e.g., epoxy siloxane), glass, modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, polytetrafluoroethylene (such as TEFLON® from Chemours), polyethylene terephthalate (PET), polycarbonate, cyclic olefins / cyclo-olefin polymers (COP) (such as ZEONOR® from Zeon), polyimides, nylon (polyamides), etc.), ceram ics / ceramic oxides, silica (i.e. , silicon dioxide (SiO )), fused silica, or silica-based materials, aluminum silicate, silicon and modified silicon (e.g., boron doped p+ silicon), silicon nitride (Si3N4), tantalum pentoxide (Ta2Os) or other tantalum oxide(s) (TaOx), hafnium oxide (HfO ), carbon, metals, inorganic glasses, or the like.

[0020] When the substrate 12 is a multi-layered structure, any of the materials described for the single layer base support may function as a base, and another layer (which has the depressions 14 defined therein) may be positioned on the base. Inthese examples, the other layer may be any material that can be etched or imprinted to form the depressions 14. Examples of the layer include inorganic oxides, such as tantalum oxide (e.g., Ta20s), aluminum oxide (e.g., AI2O3), silicon oxide (e.g., SiC>2), or hafnium oxide (e.g., HfCh), or polymeric resins, such as a polyhedral oligomeric silsesquioxane based resin (e.g., POSS® from Hybrid Plastics), a non-polyhedral oligomeric silsesquioxane epoxy resin, a poly(ethylene glycol) resin, a polyether resin (e.g., ring opened epoxies), an acrylic resin, an acrylate resin, a methacrylate resin, an amorphous fluoropolymer resin (e.g., CYTOP® from Bellex), and combinations thereof.

[0021] Many different layouts of the depressions 14 may be used regular, repeating, or non-regular patterns. In an example, the depressions 14 are disposed in a hexagonal grid for close packing and improved density. Other layouts may include, for example, rectangular layouts, triangular layouts, and so forth. In some examples, the layout or pattern can be an x-y format in rows and columns. In some other examples, the layout or pattern can be a repeating arrangement of the depressions 14 and interstitial regions 24 (i.e. , regions of the substrate surface where depressions 14 are not formed). In still other examples, the layout can be a random arrangement of the depressions 14 and the interstitial regions 24.

[0022] The layout or pattern may be characterized with respect to the density (number) of the depressions 14 in a defined area. For example, the depressions 14 may be present at a density of approximately 2 million per mm2. The density may be tuned to different densities including, for example, a density of about 100 per mm2, about 1 ,000 per mm2, about 0.1 million per mm2, about 1 million per mm2, about 2 million per mm2, about 5 million per mm2, about 10 million per mm2, about 100,000 million per mm2, or more, or less. It is to be further understood that the density can be between one of the lower values and one of the upper values selected from the ranges above, or that other densities (outside of the given ranges) may be used.

[0023] The layout or pattern of the depressions 14 may also or alternatively be characterized in terms of the average pitch, or the spacing from the center of one depression 14 to the center of an adjacent depression 14 (center-to-center spacing). The pattern can be regular, such that the coefficient of variation around the averagepitch is small, or the pattern can be non-regular in which case the coefficient of variation can be relatively large. In either case, the average pitch can be, for example, about 50 nm, about 0.15 pm, about 0.5 pm, about 1 pm, about 5 pm, about 10 pm, about 100 pm, or more or less. The average pitch for a particular pattern of depressions 14 can be between one of the lower values and one of the upper values selected from the ranges herein. In an example, the depressions 14 have a pitch (center-to-center spacing) of about 1 .5 pm. While example average pitch values have been provided, it is to be understood that other average pitch values may be used.

[0024] The size of each depression 14 is sufficient to receive the bead 16 used in the array 10. In some examples, the diameter of the bead 16 is 200 pm or less (e.g., 200 nm), the depth and diameter or length and width may be sufficient to accommodate a single bead 16. The depth can range from about 0.1 pm to about 210 pm, e.g., about 0.5 pm, about 1 pm, about 10 pm, or more, or less. The diameter or each of the length and width can range from about 0.1 pm to about 210 pm, e.g., about 0.5 pm, about 1 pm, about 10 pm, or more, or less.

[0025] In some instances, the size of the depression 14 is sufficient to physically immobilize the bead 16 in the depression 14. This is depicted in Fig. 1 B. In other instances, a capture agent may be used to immobilize the bead 16 in the depression 14. The capture agent may be any chemical, electrostatic, or hydrophilic / hydrophobic functionalization that can immobilize the bead 16 in the depression 14. One example of a capture agent is a capture primer, and the bead 16 includes a complementary primer that can hybridize to the capture primer. Another example of a capture agent is a first member of a binding pair, and the bead 16 includes a second member of the binding pair (e.g., biotin-avidin or biotin-streptavidin). Still another example of a capture agent is a material that can attract a magnetic bead. The capture agent may be selectively deposited or otherwise selectively introduced (e.g., using masking techniques) into the depressions 14 so that the interstitial regions 24 remain free of the capture agent and thus free of the beads.

[0026] As used herein, the term “bead” refers to a small body made of a rigid or semi-rigid material. The body can have a shape characterized, for example, as a sphere, oval, microsphere, or other recognized particle shape whether having regularor irregular dimensions. Example materials that are useful for beads 16 include, glass, such as modified or functionalized glass; plastic, such as acrylic, polystyrene or a copolymer of styrene and another material, polypropylene, polyethylene, polybutylene, polyurethane, or polytetrafluoroethylene (e.g., TEFLON™ from DuPont); polysaccharides or cross-linked polysaccharides, such as agarose or Sepharose; polyamide; nitrocellulose; resin; silica; silicon and modified silicon; carbon-fiber; or metal. Example beads 16 include controlled pore glass beads, paramagnetic beads, thoria sol, and Sepharose beads. In one example, the beads 16 are silica beads.

[0027] In the examples disclosed herein, at least 4% of the beads 16 in the array 10 have the sample probes 18, 20 attached thereto. In the array 10, an equal number of unmethylated probes 18 and corresponding methylated probes 20 are included, and thus the total percentage accounts for both probes 18 and 20. In some instances, all of the beads 16 in the array 10 have the sample probes 18, 20 described herein attached thereto. Thus, the array 10 may include from about 4% to 100% of sample probes 18, 20. In one example, the array 10 includes from about 4% to about 10% of the sample probes 18, 20. As will be described herein, other sample probes 22, 23, 25 may be attached to some of the beads 16 of the array 10.

[0028] In an example, the sample probes 18, 20 of a particular probe set are to capture a respective target sequence from the human genome. The target sequence can be from any chromosome, e.g., 1-22 or X.

[0029] In the array 10 disclosed herein, there are two probes 18, 20 per CpG locus of a DNA sample strand for each of the conversions described herein.

[0030] For bisulfite conversion and some enzymatic conversions (e.g., Enzymatic Methyl-seq), one of the two probes (referred to herein as the “unmethylated probe”) is for detecting the unmethylated DNA state (i.e. , C converts to U during bisulfite conversion and converts to T during amplification) of a bisulfite converted DNA sample strand, and another of the probes (referred to herein as the “methylated probe”) is for detecting the methylated DNA state (i.e., C remains C after bisulfite conversion and converts to G during amplification) of the bisulfite converted DNA sample strand. The 3’ terminus of each probe is designed to match either theprotected cytosine (methylated design) or the thymine base resulting from bisulfite conversion and whole-genome amplification (unmethylated design).

[0031] For other enzymatic conversions (e.g., using altered cytidine deaminase), one of the two probes (another example of an unmethylated probe) is for detecting the unmethylated DNA state (i.e. , C remains C) of an enzymatically converted DNA sample strand, and another of the probes (another example of a methylated probe) is for detecting the methylated DNA state (i.e., C converts to T) of the enzymatically converted DNA sample strand. The 3’ terminus of each probe is designed to match either the unmethylated cytosine or the thymine base resulting from enzymatic conversion.

[0032] In these examples, the probes 18, 20 in a probe set are based on single nucleotide polymorphisms (SNPs) found in either a top strand sequence or a bottom strand sequence at a high minor allele frequency. The following may be used to designate the strands. When the A or T in a first unambiguous pair is on the 5’ side of the CpG, then the sequence is designated as the top strand sequence. When the A or T in the first unambiguous pair is on the 3’ side of the CpG, then the sequence is designated as the bottom strand sequence.

[0033] The SNP in the top or bottom strand sequence is adjacent to a CpG site per one of following rules: i) in the top strand sequence, the reference of the SNP is [G] followed by [CG] (in the 5’->3’ direction), ii) in the bottom strand sequence, the reference of the SNP is [G] followed by [N] (where N is A, T, C, or G) and is preceded by [CG] (in the 5’— >3’ direction), or iii) in the bottom strand sequence, the reference of the SNP is [C] followed by [G] and is preceded by [CG] (in the 5’— >3’ direction).

[0034] When the probes 18, 20 are based on the top strand sequence, the top strand sequence contains a high minor allele frequency single nucleotide polymorphism where the reference is [G], which is followed by [CG] (in the 5’— >3’ direction). The top strand sequence is bisulfite converted or enzymatically converted, and the probes are then designed from the methylated version of the bisulfite or enzymatically converted top strand sequence and the unmethylated version of the bisulfite or enzymatically converted top strand sequence. An example of the top strand sequence (original TOP), the methylated version of the bisulfite converted topstrand sequence (TCM) and the unmethylated version of the bisulfite converted top strand sequence (TCU) are shown in Fig. 2. The arrows labeled 18 and 20 illustrate the respective sequences of TCU and TCM from which the probes 18 and 20 are designed.

[0035] In one example when the probes 18, 20 are based on the bottom strand sequence, the bottom strand sequence may contain a high minor allele frequency single nucleotide polymorphism where the reference is [C], which is followed by [G] and is preceded by [CG]. The bottom strand sequence is bisulfite or enzymatically converted, and the probes are then designed from the methylated version of the bisulfite or enzymatically converted bottom strand sequence and the unmethylated version of the bisulfite or enzymatically converted bottom strand sequence. An example of the bottom strand sequence (original BOT), the methylated version of the bisulfite converted bottom strand sequence (BCM) and the unmethylated version of the bisulfite converted bottom strand sequence (BCU) are shown in Fig. 3. The arrows labeled 18 and 20 illustrate the respective sequences of BCU and BCM from which the probes 18 and 20 are designed.

[0036] In another example when the probes 18, 20 are based on the bottom strand sequence, the bottom strand sequence may contain a high minor allele frequency single nucleotide polymorphism where the reference is [G], which is followed by [N] (where N is A, T, C, or G) and is preceded by [CG]. The bottom strand sequence is bisulfite converted or enzymatically converted, and the probes are then designed from the methylated version of the bisulfite or enzymatically converted bottom strand sequence and the unmethylated version of the bisulfite or enzymatically converted bottom strand sequence. An example of the bottom strand sequence (original BOT), the methylated version of the bisulfite converted bottom strand sequence (BCM) and the unmethylated version of the bisulfite converted bottom strand sequence (BCU) are shown in Fig. 4. The arrows labeled 18 and 20 illustrate the respective sequences of BCU and BCM from which the probes 18 and 20 are designed.

[0037] Tables 1 -23 illustrate several examples of sets of probes 18, 20, which are designed in accordance with the rules set forth herein from human chromosomes1-22 and X and using bisulfite conversion. Each row of the tables identifies an unmethlyated probe by its SEQ. ID. NO. as set forth in the sequence listing filed herewith, the corresponding methylated probe by its SEQ. ID. NO. as set forth in the sequence listing filed herewith, the position of the reference nucleotide of the SNP, and whether the [CG] preceded or followed the reference position.

[0038] Each of the sample probes 18, 20 is a 50-mer, which is based on the assumption that methylation is regionally correlated within a 50 base pair (bp) span.

[0039] Each of the probes 18, 20 may also include a barcode (decoder) portion at its 5’ end. The barcode portion is a nucleotide sequence that may be used to distinguish individual beads 16. The barcode can be added to the probe 18, 20 by methods that physically link or bond the decoder to the probe molecules, e.g., by ligation or transposition through polymerase, endonuclease, transposases, etc.

[0040] In the examples disclosed herein, all of the probes 18 and 20 are attached to respective bead 16. While a single sample probe 18 or 20 is shown attached to each bead 16, it is to be understood that each bead is coated with multiple copies of the respective probes 18, 20. The 5’ terminus of each probe 18, 20 may be modified to allow a coupling reaction with a functional group at or introduced to a surface of the beads 16. An example of a 5’ terminal group is biotin.

[0041] The surface of the beads 16 can include physical alterations to attach the probes 18, 20. For example, the surface of a bead 16 can be modified to contain chemically modified sites that are useful for attaching, either-covalently or non- covalently, the probes 18, 20. The bead 16 surface may include chemical functional groups including amino groups, carboxy groups, oxo groups and thiol groups, that can be used to covalently attach corresponding reactive 5’ terminal groups of the probes 18, 20. In one example, the beads 16 are coated with streptavidin (to non-covalently attached a biotinylated probe 18, 20.

[0042] The probes 18, 20 can be attached by sequential addition of monomeric units to synthesize the probes in situ. Probes 18, 20 can alternatively be synthesized, and then attached using any of a variety of methods known in the art including printing techniques (e.g., ink-jet printing), a spotting technique, a photolithographic synthesis, or printing methods that utilize a mask.

[0043] The array 10 may be included in a methylation detection kit. One example of the kit includes any example of the array 10 disclosed herein and a sodiumbisulfite solution that can be used in the bisulfite conversion of the DNA sample that is to be used with the array 10.

[0044] Another example of the kit includes an enzymatic methylation conversion mix that can be used in the enzymatic conversion of the DNA sample that is to be used with the array 10.

[0045] The enzymatic methylation conversion mix includes a liquid carrier and an altered cytidine deaminase. Examples of this mix are described in International Patent Application No. PCT / US2023 / 017846, entitled, “Altered Cytidine Deaminases and Methods of Use” (published as WO 2023 / 196572) and International Patent Application No. PCT / IB2023 / 059798, entitled, “Helicase-Cytidine Deaminase Complexes and Methods of Use,” each of which is incorporated herein by reference in its entirety.

[0046] The liquid carrier in the enzymatic methylation conversion mix may be a buffer having a pH lower than 7 (e.g., ranging from 5.1 to 6.5). Examples of suitable buffers include, but are not limited to: a citrate buffer, a sodium acetate buffer, Bis TrisPropane HC1 , and Tris-HCI Tris. Examples of other buffers include, but are not limited to, Bicine, DIPSO (3-[N,N-Bis(2-hydroxyethylamino)-2-hydroxy-1 -propanesulfonic acid), glycylglycine, HEPES (2-[4-(2-hydroxyethyl)piperazin-1 -yl]ethanesulfonic acid), imidazole, malonate, MES (2-(N-morpholino)ethanesulfonic acid), MOPS (3-(N- morpholino)propanesulfonic acid), phosphate, PIPES (1 ,4-Piperazinediethanesulfonic acid), SPG (succinic acid, sodium dihydrogen phosphate, and glycine in the molar ratio 2:7:7), succinate, TAPS (N-[Tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid), TAPSO (2-Hydroxy-3-[tris(hydroxymethyl)methylamino]-1 -propanesulfonic acid), trincine. In some examples, a reducing agent such as dithiothreitol (DTT) can be present. In some examples, a divalent cation is not included.

[0047] The enzymatic methylation conversion also includes the altered cytidine deaminase.

[0048] In the examples set forth herein, the type of altered cytidine deaminase that is used preferentially deaminates 5mC instead of C (i.e. , converts 5mC to T at a greater rate than converting C to U) and thus has cytosine-defective deaminase activity or 5mC-enhanced or 5mC-selecting deaminase activity. In one example, thealtered cytidine deaminase having cytosine-defective deaminase activity includes a substitution mutation at a position functionally equivalent to tyrosine at position 130 (Y130) in a member of the APOBEC3A subfamily (for instance, SEQ. ID. NO. 29947). This substitution mutation can be a mutation to alanine (A), glycine (G), phenylalanine (F), histidine (H), glutamine (Q), methionine (M), asparagine (N), lysine (K), valine (V), aspartic acid (D), glutamic acid (E), serine (S), cysteine (C), proline (P), or threonine (T). For example, the altered cytidine deaminase can be SEQ. ID. NO. 29948, wherein X is selected from A, G, F, H, Q, M, N, K, V, D, E, S, C, P or T (and is not Y), or can comprise SEQ. ID. NO. 29949, wherein X is selected from A, G, F, H, Q, M, N, K, V, D, E, S, C, P or T (and is not Y). In specific examples of SEQ. ID. NO. 29948 or SEQ. ID. NO. 29949, X is A or L. As one specific example, the substitution mutation at a position functionally equivalent to Y130 is a mutation to alanine (A), (e.g., SEQ. ID. NO. 29950). Specific examples of altered cytidine deaminases having increased activity and preferentially acting on 5mC compared to cytosine include SEQ. ID. NO. 29950 or a sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity to SEQ. ID. NO. 29950 and including Y130A.

[0049] The altered cytidine deaminase having cytosine-defective deaminase activity optionally includes a second substitution mutation at a position two, three, four, or five amino acids on the C-terminal side of the Y130 position, or functionally equivalent to the Y130 position. In one example, the second mutation is a tyrosine (Y), tryptophan (W), cysteine (C), histidine (H), or phenylalanine (F) at a position two, three, four, or five amino acids on the C-terminal side of the Y130 position, or functionally equivalent to the Y130 position. In one example, the second mutation is at a position functionally equivalent to tyrosine at position 132 (Y132) in a member of the APOBEC3A subfamily (for instance, SEQ. ID. NO. 29947). An APOBEC protein, such as an APOBEC3A protein, containing substitution mutations at both the first site, a position functionally equivalent to Y130, and the second site, at a position two, three, four, or five amino acids on the C-terminal side of the Y130 position, increases the preferential activity to act on 5mC compared to the same APOBEC protein, such as an APOBEC3A protein, containing one substitution mutation at Y130. In one example, the substitution mutation at the second position is an amino acid having a positivelycharged side chain and selected from arginine (R), histidine (H), lysine (L), or a polar side chain selected from glutamine (Q). As a specific example, the substitution mutation at the second position is histidine (H), such as Y132 to histidine. The double mutant containing both first and second mutations can be any substitution mutation at a position functionally equivalent to Y130 described herein and any second substitution mutation at a position two, three, four, or five amino acids on the C- terminal side of the Y130 position described herein, in any combination. For example, the altered cytidine deaminase can be SEQ. ID. NO.: 29947, 29951 , or 29950 and have a substitution at Y130 and Y132, or the position functionally equivalent to Y130 and Y132 as described herein. One example of an altered cytidine deaminase is SEQ. ID. NO. 29952 including Y130X and Y132X, where Y130X is selected from (A), (L), or (W) (preferably (A)), and Y132X is selected from (R), (H), (L), or (Q), preferably (H). This encompasses examples including Y130A and Y132R, Y130A and Y132H, Y130A and Y132L, Y130A and Y132Q, Y130L and Y132R, Y130L and Y132H, Y130L and Y132L, Y130L and Y132Q, Y130W and Y132R, Y130W and Y132H, Y130W and Y132L, Y130W and Y130Q, or any suitable combinations therein. In one example, the double mutant includes substitution mutations Y130A and Y132H. Specific examples of altered cytidine deaminases having both substitution mutations and preferentially acting on 5mC compared to the APOBEC protein having just the single substitution mutation at cytosine include SEQ. ID. NO. 29953 or a sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity to SEQ. ID. NO. 5281 and including Y130A and Y132H.

[0050] The enzymatic methylation conversion mix includes the modified cytidine deaminase at a concentration from at least 0.05 micromolar (pM) (i.e. , 50 nM) to no greater than 5 pM. As examples instances, the concentration of the enzyme can be at least 0.5 pM, or at least 1 pM, or at least 2DpM, or at least 3 pM, or at least 4 pM, or 5 pM, and / or no greater than 5 pM, or no greater than 4 pM, or no greater than 3DpM, or no greater than 2 pM, or no greater than 1 pM. In some specific examples, the concentration of the enzyme can be about 0.4 pM, or about 0.5 pM, or about 0.8 pM.

[0051] One example of the array 10 includes at least 100 different probes 18 and / or 20, where at least 50 of the at least 100 different probes 18 are unmethylatedprobes selected from the group consisting of SEQ. ID. NO. 1 through SEQ. ID. NO. 14,973; and at least 50 other of the at least 100 different probes are methylated probes selected from the group consisting of SEQ. ID. NO. 14,974 through SEQ. ID. NO. 29,946. When this example array includes an unmethylated probe 18 and its corresponding methylated probe 20, the array may be used for methylation detection, background normalization, and genotyping as described herein. Alternatively, the at least 100 different probes 18 and / or 20 may be selected for target capture or enrichment. In these examples, it is desirable to narrow down the number of sample (library) fragments that are subsequently tested for methylation or are genotyped, and thus probe sequences are specifically selected to capture the sample fragments of interest. In these types of panels, the sample fragments hybridize to a complementary probe, and then the unattached sample fragments are removed. The hybridized sample fragments are then dehybridized and transmitted to another array for methylation detection or genotyping.

[0052] The target capture panel may be a separate compartment that is selectively fluidly connected to, and upstream of, the array 10. The target capture panel may include preselected probes 18, 20 attached to a solid surface, e.g., beads 16 in the depressions 14 of the substrate 24 or the substrate 24 itself, for capturing specific sample (library) fragments. The compartment may also be selectively fluidly connected to a waste container to receive the unattached sample fragments before the captured sample fragments are dehybridized and directed toward the array 10. Selective fluid connections may be achieved using valves and fluidic lines between the various components (e.g., compartment, array 10, waste container).

[0053] The enrichment panel may be a separate solution that contains the preselected probes 18, 20. In this example, the probes 18, 20 may be attached to magnetic beads for ease of separation of the unattached sample fragments from the captured sample fragments, and of the dehybridzed sample fragments from the beads 16 and probes 18, 20. With this example, the dehybridzed sample fragments may be introduced into the array 10.

[0054] As mentioned, the sample probes 18, 20 disclosed herein may make up less than 100% of the sample probes in an array 10. Other sample probes 22 or 23and 25 may be used in combination with the probes 18, 20. The probes 22 or 23 and 25 can be categorized as two different types.

[0055] The first type includes two probes 23, 25 (unmethylated and methylated) per CpG locus of the DNA sample strands. In one example, the 3’ terminus of each of these probes 23, 25 is designed to match either the protected cytosine (methylated design) or the thymine base resulting from bisulfite conversion and whole-genome amplification (unmethylated design). In another example, the 3’ terminus of each of these probes 23, 25 is designed to match either the unmethylated cytosine (unmethylated design) or the thymine base resulting from enzymatic conversion and whole-genome amplification (methylated design).

[0056] The second type includes a single probe 22 per CpG locus. In one example, the 3’ terminus of this probe 22 complements the base directly upstream of the query site, and the single base extension results in the addition of a labeled G or A base, complementary to either the methylated C or the T (unmethylated state) of the bisulfite converted DNA sample strand. In another example, the 3’ terminus of this probe 22 complements the base directly upstream of the query site, and the single base extension results in the addition of a labeled A or G base, complementary to either the T (methylated state) or the unmethylated C of the enzymatically converted DNA sample strand.

[0057] The sample probes 22 or 23 and 25 can be based on the original DNA target strands, the bisulfite converted DNA target strands, and / or the complements of the bisulfite converted DNA target strands. The sample probes 22 or 23 and 25 can alternatively be based on the original DNA target strands, the enzymatically converted DNA target strands, and / or the complements of the enzymatically converted DNA target strands. Fig. 5 illustrates one example of design principles that can be used for the sample probes 22 and 23, 25. In this example, two probes 23, 25 are used for targets having greater than 4 CpG sites, including a completely methylated probe (having a G nucleotide that complements the C position of each CpG site) and completely unmethylated probe (having an A nucleotide that complements the U that is expected to result from bisulfite conversion of each of the C positions of a CpG site) as shown in Fig. 5. In contrast, a single probe 22 (labeled “degenerate probe” in Fig.5) is used for targets having 4 or fewer CpG sites (the probe includes degenerate nucleotide R, complementary to U or C, at the C position of each CpG site). It is to be understood that other design principles may be used for the other probes 22 or 23 and 25, such as those described in U.S. Patent No. 8,150,626, which is incorporated herein by reference in its entirety.

[0058] Similar design principles can be used for the sample probes 22 or 23 and 25 for enzymatically converted samples. An example of an enzymatically converted sample including 4 CpG sites is shown in Fig. 6.

[0059] Examples of sample probes for this enzymatically converted sample include a completely methylated probe (having an A nucleotide that complements the T that is expected to result from enzymatic conversion of each of the C positions of a CpG site) and a completely unmethylated probe (having a G nucleotide that complements the C position of each CpG site). Alternatively, a single probe is used for targets having 4 or fewer CpG sites (the probe includes degenerate nucleotide Y, complementary to G or A, at the C position of each CpG site).

[0060] In the array 10 disclosed herein, from 4% to 100% of the beads 16 in the array 10 have the sample probes 18, 20 attached thereto. As such, from 0% to 96% of the beads 16 in the array 10 have the sample probes 22, 23, 25 attached thereto.When the sample probes are present, from about 16% to about 26% of the sample probes 22, 23, 25 are of the first type (i.e. , sample probes 23 and 25), and from about 70% to about 80% of the sample probes 22, 23, 25 are of the second type (i.e., sample probes 22).

[0061] An example of the method for using the array 10 includes generating a bisulfite converted and amplified DNA sample; and performing a methylation assay using the methylation detection array 10.

[0062] For the bisulfite conversion, DNA is first denatured (made singlestranded) and then treated with sodium bisulfite. The converted DNA is also amplified. The methylation assay enables hybridization of the converted and amplified DNA sample strands to the sample probes 18, 20 and the other sample probes 22, 23, 25.

[0063] An extension reaction is performed at the 3’ ends of the probes 18, 20, 22, 23, 25. With any of the sample probes 18, 20, 23, 25 disclosed herein, whenbisulfite converted DNA target strands - with the unmethylated CpG sites - hybridize to the unmethylated probe 18, 23, and when the bisulfite converted DNA target strands - with the methylated CpG sites - hybridize to the methylated probe 20, 25, single-base extension is enabled. With the sample probe 22, the single base extension results in the addition of a labeled G or A base, complementary to either the methylated C or unmethylated T of the bisulfite converted DNA sample strand. As described in more detail below, single-base extension is performed with labeled nucleotides, which are subsequently stained with a fluorescent reagent and a polymerase. The labeled nucleotides may include T tagged with red channel labels and G tagged with green channel labels. The use of opposed color channels enables one to interpret the data correctly. The level of methylation for the interrogated locus can be determined by calculating the ratio of the fluorescent signals from the methylated versus unmethylated sites. In contrast, a mismatched base at the query site will inhibit extension. Thus, the probes 18, 20 or 23, 25 of a single probe set enable correct methylation calls to be identified for the corresponding target strand.

[0064] Another example of the method for using the array 10 includes generating an enzymatically converted and amplified DNA sample; and performing a methylation assay using the methylation detection array 10.

[0065] For one example of the enzymatic conversion, DNA is first denatured (made single-stranded) and then treated with the enzymatic methylation conversion mix disclosed herein (including a liquid carrier and an altered cytidine deaminase). During exposure of the DNA to the enzymatic methylation conversion mix, the conversion of 5-methylcytosine (5mC) to thymidine (T) by deamination takes place at a greater rate than conversion of cytosine (C) to uracil (II) by deamination, resulting in a converted single-stranded DNA fragment (see the example shown in Fig. 3). The converted DNA is also amplified. The methylation assay enables hybridization of the converted and amplified DNA sample strands to the sample probes 18, 20 and the other sample probes 22, 23, 25.

[0066] An extension reaction is performed at the 3’ ends of the probes 18, 20, 22, 23, 25. Similar to the sample probes 18, 20, 23, 25 for bisulfite conversion, when the enzymatically converted DNA target strands - with the unmethylated CpG sites -hybridize to the unmethylated probe, and when the enzymatically converted DNA target strands - with the methylated CpG sites - hybridize to the methylated probe, single-base extension is enabled. Single-base extension is performed with labeled nucleotides, which are subsequently stained with a fluorescent reagent. The level of methylation for the interrogated locus can be determined by calculating the ratio of the fluorescent signals from the methylated versus unmethylated sites. In contrast, a mismatched base at the query site will inhibit extension. Similar to the sample probe 22 for bisulfite conversion, the 3’ terminus of the sample probe 22 complements the base directly upstream of the query site, and the single base extension results in the addition of a labeled A or G base, complementary to either the T (methylated state) or the unmethylated C of the enzymatically converted DNA sample strand.

[0067] As mentioned, regardless of the type of conversion that is formed, the conversion is followed by an extension reaction. For the extension reaction, a mixture containing nucleotides and a polymerase is introduced into the array 10. The nucleotides include the following bases: adenine, cytosine, guanine (tagged with green channel label) and thymine (tagged with a red channel label). Any polymerase that can accept the nucleotide, and that can successfully incorporate the base of the nucleotide at the 3’ end of the probe 18, 20, 22, 23, 25 may be used. Example polymerases include those polymerases from family A, such as Bsu Polymerase, Bst Polymerase, Taq Polymerase, T7 Polymerase, and many others; polymerases from families B and B2, such as Phi29 polymerase and other highly processive polymerases (family B2), Pfu Polymerase (family B), KOD Polymerase (family B), 9oN (family B), and many others; polymerases from family C, such as Escherichia coli DNA Pol III, and many others, polymerases from family D, such as Pyrococcus furiosus DNA Pol II, and many others; polymerases from family X, such as DNA Pol p, DNA Pol p, DNA Pol o, and many others. The nucleotide mixture may also include a liquid carrier, such as water and / or an ionic salt buffer fluid, e.g., saline citrate at m illi-molar to molar concentrations, sodium chloride, potassium chloride, phosphate buffered saline, etc., and other buffers, such as tris(hydroxymethyl)aminomethane (TRIS) or (4- (2-hydroxyethyl)-1 -piperazineethanesulfonic acid) (HEPES). The liquid carrier mayalso include catalytic metal(s) intended for the extension reaction, such as Mg2+, Mn2+, etc. A single catalytic metal or a combination of catalytic metals may be used, and the total amount may range from about 0.01 mM to about 100 mM.

[0068] The temperature of the array 10 may be adjusted to initiate the extension reaction. Example temperatures range from about 20°C to about 70°C. The polymerase enables the extension of the 3’ end of the probe 18, 20. As described, the first extension reaction and the color signal data obtained from this reaction enables one to determine the methylation status and the genotyping status.

[0069] The data derived from this extension reaction (or lack thereof) may be used as a discovery tool in order to determine the ethnicity of the individual whose sample is being tested, to identify new single nucleotide polymorphisms in the particular sample, for sample finger printing, sample tracking, or the like.

[0070] The sample probes 18, 20 described herein can also be used for background normalization. Because the sample probes 18, 20 are part of a probe set, the out of band channel for the respective probes 18, 20 can be used for background normalization. In one example, all fluorescence from probes 18, 20 at the wavelength of the opposite fluorophore — that which is not the extension base — can be used to estimate non-specific fluorescence across the array 10. In another example, the pOOBAH (P-value with OOB probes for Array Hybridization) method is used, where detection P-value is calculated using an empirical cumulative distribution function (implemented in the ecdf function in R stats package) derived from the OOB signal from all of the probes 18, 20. In still another example, stratified quantile normalization (preprocessQuantile) is used.

[0071] Some examples presented herein describe analysis of bisulfite converted samples. In bisulfite methods, DNA is chemically treated with sodium bisulfite, which results in the conversion of unmethylated cytosines to uracils, and the resulting uracils are ultimately detected as thymines. In contrast, the modified cytosines, 5mC and 5hmC, are resistant to bisulfite conversion, and are detected as cytosines. Other examples presented herein use a modified cytidine deaminase enzyme, engineered to selectively deaminate only 5mC and 5hmC, while unmethylated cytosines are not converted. This modified cytidine deaminase method results in conversion of 5mCand 5hmC to uracils, and the resulting uracils are ultimately detected as thymines. In contrast, unmethylated cytosines are resistant to the deaminase conversion, and are detected as cytosines. Use of modified cytidine deaminase enzymes has been described in PCT application PCT / US2023 / 17846, filed April 7, 2023 and titled “Altered Cytidine Deaminases and Methods of Use”, the content of which is incorporated herein by reference in its entirety. One of ordinary skill in the art will recognize that where this modified cytidine deaminase conversion method is utilized to generate converted DNA, the probes and primers described herein can be modified accordingly to reflect methylated targets where methylated cytosines are converted and detected as thymine (C to T conversion) and unmethylated cytosines are not converted and detected as cytosine.

[0072] It is to be understood by one of ordinary skill in the art that other methylation conversion methods can also be used to generate converted DNA for use in the methods and examples presented herein. For example, an alternative to bisulfite conversion is Enzymatic Methyl-seq (EM-seq). EM-seq has been described in neb.com / - / media / nebus / files / manuals / manuale7120. pdf, the content of which is incorporated herein by reference in its entirety. Briefly, EM-seq conversion uses an enzymatic method which results in the conversion of unmethylated cytosines to uracils, and the resulting uracils are ultimately detected as thymines. In contrast, the modified cytosines, 5mC and 5hmC, are resistant to the enzymatic conversion, and are detected as cytosines. Another example of an alternative method to bisulfite conversion is TET-assisted pyridine borane sequencing (TAPS). TAPS has been described in Liu, et al. Bi sulfite-free direct detection of 5-methylcytosine and 5- hydroxymethylcytosine at base resolution. Nat Biotechnol. 2019 Apr;37(4):424-429. doi:10.1038 / s41587-019-0041 -2, the content of which is incorporated herein by reference in its entirety. TAPS results in conversion of 5mC and 5hmC to uracils, and the resulting uracils are ultimately detected as thymines. In contrast, unmethylated cytosines are resistant to the TAPS conversion, and are detected as cytosines. One of ordinary skill in the art will recognize that where TAPS conversion is utilized to generate converted DNA, the probes and primers described herein can be modified accordingly to reflect methylated targets where methylated cytosines are convertedand detected as thymine (C to T conversion) and unmethylated cytosines are not converted and sequenced as cytosine

[0073] The singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[0074] The terms comprising, including, containing and various forms of these terms are synonymous with each other and are meant to be equally broad.

[0075] The terms top, bottom, lower, upper, on, etc. are used herein to describe the flow cell and / or the various components of the flow cell. It is to be understood that these directional terms are not meant to imply a specific orientation, but are used to designate relative orientation between components. The use of directional terms should not be interpreted to limit the examples disclosed herein to any specific orientation(s).

[0076] The terms first, second, etc. also are not meant to imply a specific orientation or order, but rather are used to distinguish one component from another.

[0077] It is to be understood that the ranges provided herein include the stated range and any value or sub-range within the stated range, as if such values or subranges were explicitly recited. For example, a range of about 400 nm to about 1 pm (1000 nm), should be interpreted to include not only the explicitly recited limits of about 400 nm to about 1 pm, but also to include individual values, such as about 708 nm, about 945.5 nm, etc., and sub-ranges, such as from about 425 nm to about 825 nm, from about 550 nm to about 940 nm, etc.

[0078] Furthermore, when “about” and / or “substantially” are / is utilized to describe a value, they are meant to encompass minor variations (up to + / - 10%) from the stated value.

[0079] Reference throughout the specification to “one example”, “another example”, “an example”, and so forth, means that a particular element (e.g., feature, structure, and / or characteristic) described in connection withO the example is included in at least one example described herein, and may or may not be present in other examples. In addition, it is to be understood that the described elements for any example may be combined in any suitable manner in the various examples unless the context clearly dictates otherwise.

[0080] While several examples have been described in detail, it is to be understood that the disclosed examples may be modified. Therefore, the foregoing description is to be considered non-limiting.

Claims

What is claimed is:

1. A methylation detection array, comprising: a substrate having a plurality of depressions defined therein; a plurality of beads, each of the plurality of beads positioned within one of the plurality of depressions; and a plurality of sample probes respectively attached to the plurality of beads, the plurality of sample probes including a plurality of probe sets, wherein: each of the plurality of probe sets includes an unmethylated probe and a corresponding methylated probe; and the unmethylated probe and the corresponding methylated probe are: i) each based on a bisulfite or enzymatically converted top strand sequence of a corresponding top strand sequence that contains a high minor allele frequency single nucleotide polymorphism where a reference is [G] followed by [CG]; or ii) each based on a bisulfite or enzymatically converted bottom strand sequence of a corresponding bottom strand sequence that contains a high minor allele frequency single nucleotide polymorphism where a reference is [G] followed by [N] and preceded by [CG]; or iii) each based on a bisulfite or enzymatically converted bottom strand sequence of a corresponding bottom strand sequence that contains a high minor allele frequency single nucleotide polymorphism where a reference is [C] followed by [G] and preceded by [CG].

2. The methylation detection array as defined in claim 1 , wherein: at least one of the plurality of probe sets is based on chromosome 1 from a human genome; the unmethylated probe is selected from the group consisting of SEQ. ID. NO. 1 through SEQ. ID. NO. 1 ,167; and the corresponding methylated probe is selected from the group consisting of SEQ. ID. NO. 14,974 through SEQ. ID. NO. 16,140.

3. The methylation detection array as defined in any one of claim 1 or claim 2, wherein: at least one of the plurality of probe sets is based on chromosome 2 from a human genome; the unmethylated probe is selected from the group consisting of SEQ. ID. NO.1 ,168 through SEQ. ID. NO. 2,144; and the corresponding methylated probe is selected from the group consisting of SEQ. ID. NO. 16,141 through SEQ. ID. NO. 17,117.

4. The methylation detection array as defined in any one of claim 1 through claim 3, wherein: at least one of the plurality of probe sets is based on chromosome 3 from a human genome; the unmethylated probe is selected from the group consisting of SEQ. ID. NO.2,145 through SEQ. ID. NO. 2,891 ; and the corresponding methylated probe is selected from the group consisting of SEQ. ID. NO. 17,118 through SEQ. ID. NO. 17,864.

5. The methylation detection array as defined in any one of claim 1 through claim 4, wherein: at least one of the plurality of probe sets is based on chromosome 4 from a human genome; the unmethylated probe is selected from the group consisting of SEQ. ID. NO.2,892 through SEQ. ID. NO. 3,720; and the corresponding methylated probe is selected from the group consisting of SEQ. ID. NO. 17,865 through SEQ. ID. NO. 18,693.

6. The methylation detection array as defined in any one of claim 1 through claim 5, wherein: at least one of the plurality of probe sets is based on chromosome 5 from a human genome;the unmethylated probe is selected from the group consisting of SEQ. ID. NO.3,721 through SEQ. ID. NO. 4,417; and the corresponding methylated probe is selected from the group consisting of SEQ. ID. NO. 18,694 through SEQ. ID. NO. 19,390.

7. The methylation detection array as defined in any one of claim 1 through claim 6, wherein: at least one of the plurality of probe sets is based on chromosome 6 from a human genome; the unmethylated probe is selected from the group consisting of SEQ. ID. NO. 4,418 through SEQ. ID. NO. 5,276; and the corresponding methylated probe is selected from the group consisting of SEQ. ID. NO. 19,391 through SEQ. ID. NO. 20,249.

8. The methylation detection array as defined in any one of claim 1 through claim 7, wherein: at least one of the plurality of probe sets is based on chromosome 7 from a human genome; the unmethylated probe is selected from the group consisting of SEQ. ID. NO. 5,277 through SEQ. ID. NO. 6,150; and the corresponding methylated probe is selected from the group consisting of SEQ. ID. NO. 20,250 through SEQ. ID. NO. 21 ,123.

9. The methylation detection array as defined in any one of claim 1 through claim 8, wherein: at least one of the plurality of probe sets is based on chromosome 8 from a human genome; the unmethylated probe is selected from the group consisting of SEQ. ID. NO. 6,151 through SEQ. ID. NO. 6,906; and the corresponding methylated probe is selected from the group consisting of SEQ. ID. NO. 21 ,124 through SEQ. ID. NO. 21 ,879.

10. The methylation detection array as defined in any one of claim 1 through claim 9, wherein: at least one of the plurality of probe sets is based on chromosome 9 from a human genome; the unmethylated probe is selected from the group consisting of SEQ. ID. NO.6,907 through SEQ. ID. NO. 7,566; and the corresponding methylated probe is selected from the group consisting of SEQ. ID. NO. 21 ,880 through SEQ. ID. NO. 22,539.11 . The methylation detection array as defined in any one of claim 1 through claim 10, wherein: at least one of the plurality of probe sets is based on chromosome 10 from a human genome; the unmethylated probe is selected from the group consisting of SEQ. ID. NO.7,567 through SEQ. ID. NO. 8,359; and the corresponding methylated probe is selected from the group consisting of SEQ. ID. NO. 22,540 through SEQ. ID. NO. 23,332.

12. The methylation detection array as defined in any one of claim 1 through claim 11 , wherein: at least one of the plurality of probe sets is based on chromosome 11 from a human genome; the unmethylated probe is selected from the group consisting of SEQ. ID. NO.8,360 through SEQ. ID. NO. 8,993; and the corresponding methylated probe is selected from the group consisting of SEQ. ID. NO. 23,333 through SEQ. ID. NO. 23,966.

13. The methylation detection array as defined in any one of claim 1 through claim 12, wherein:at least one of the plurality of probe sets is based on chromosome 12 from a human genome; the unmethylated probe is selected from the group consisting of SEQ. ID. NO. 8,994 through SEQ. ID. NO. 9,704; and the corresponding methylated probe is selected from the group consisting of SEQ. ID. NO. 23,967 through SEQ. ID. NO. 24,677.

14. The methylation detection array as defined in any one of claim 1 through claim 13, wherein: at least one of the plurality of probe sets is based on chromosome 13 from a human genome; the unmethylated probe is selected from the group consisting of SEQ. ID. NO.9,705 through SEQ. ID. NO. 10,145; and the corresponding methylated probe is selected from the group consisting of SEQ. ID. NO. 24,678 through SEQ. ID. NO. 25,118.

15. The methylation detection array as defined in any one of claim 1 through claim 14, wherein: at least one of the plurality of probe sets is based on chromosome 14 from a human genome; the unmethylated probe is selected from the group consisting of SEQ. ID. NO. 10,146 through SEQ. ID. NO. 10,588; and the corresponding methylated probe is selected from the group consisting of SEQ. ID. NO. 25,119 through SEQ. ID. NO. 25,561.

16. The methylation detection array as defined in any one of claim 1 through claim 15, wherein: at least one of the plurality of probe sets is based on chromosome 15 from a human genome; the unmethylated probe is selected from the group consisting of SEQ. ID. NO. 10,589 through SEQ. ID. NO. 11 ,022; andthe corresponding methylated probe is selected from the group consisting of SEQ. ID. NO. 25,562 through SEQ. ID. NO. 25,995.

17. The methylation detection array as defined in any one of claim 1 through claim 16, wherein: at least one of the plurality of probe sets is based on chromosome 16 from a human genome; the unmethylated probe is selected from the group consisting of SEQ. ID. NO. 11 ,023 through SEQ. ID. NO. 11 ,666; and the corresponding methylated probe is selected from the group consisting of SEQ. ID. NO. 25,996 through SEQ. ID. NO. 26,639.

18. The methylation detection array as defined in any one of claim 1 through claim 17, wherein: at least one of the plurality of probe sets is based on chromosome 17 from a human genome; the unmethylated probe is selected from the group consisting of SEQ. ID. NO. 11 ,667 through SEQ. ID. NO. 12,350; and the corresponding methylated probe is selected from the group consisting of SEQ. ID. NO. 26,640 through SEQ. ID. NO. 27,323.

19. The methylation detection array as defined in any one of claim 1 through claim 18, wherein: at least one of the plurality of probe sets is based on chromosome 18 from a human genome; the unmethylated probe is selected from the group consisting of SEQ. ID. NO. 12,351 through SEQ. ID. NO. 12,727; and the corresponding methylated probe is selected from the group consisting of SEQ. ID. NO. 27,324 through SEQ. ID. NO. 27,700.

20. The methylation detection array as defined in any one of claim 1 through claim 19, wherein: at least one of the plurality of probe sets is based on chromosome 19 from a human genome; the unmethylated probe is selected from the group consisting of SEQ. ID. NO. 12,728 through SEQ. ID. NO. 13,496; and the corresponding methylated probe is selected from the group consisting of SEQ. ID. NO. 27,701 through SEQ. ID. NO. 28,469.

21. The methylation detection array as defined in any one of claim 1 through claim 20, wherein: at least one of the plurality of probe sets is based on chromosome 20 from a human genome; the unmethylated probe is selected from the group consisting of SEQ. ID. NO. 13,497 through SEQ. ID. NO. 13,918; and the corresponding methylated probe is selected from the group consisting of SEQ. ID. NO. 28,470 through SEQ. ID. NO. 28,891.

22. The methylation detection array as defined in any one of claim 1 through claim 21 , wherein: at least one of the plurality of probe sets is based on chromosome 21 from a human genome; the unmethylated probe is selected from the group consisting of SEQ. ID. NO. 13,919 through SEQ. ID. NO. 14,155; and the corresponding methylated probe is selected from the group consisting of SEQ. ID. NO. 28,892 through SEQ. ID. NO. 29,128.

23. The methylation detection array as defined in any one of claim 1 through claim 22, wherein: at least one of the plurality of probe sets is based on chromosome 22 from a human genome;the unmethylated probe is selected from the group consisting of SEQ. ID. NO. 14,156 through SEQ. ID. NO. 14,567; and the corresponding methylated probe is selected from the group consisting of SEQ. ID. NO. 29,129 through SEQ. ID. NO. 29,540.

24. The methylation detection array as defined in any one of claim 1 through claim 23, wherein: at least one of the plurality of probe sets is based on chromosome X from a human genome; the unmethylated probe is selected from the group consisting of SEQ. ID. NO. 14,568 through SEQ. ID. NO. 14,973; and the corresponding methylated probe is selected from the group consisting of SEQ. ID. NO. 29,541 through SEQ. ID. NO. 29,946.

25. The methylation detection array as defined in claim 1 wherein: the sample probes include at least 100 different probes; at least 50 of the at least 100 different probes are unmethylated probes selected from the group consisting of SEQ. ID. NO. 1 through SEQ. ID. NO. 14,973; and at least 50 other of the at least 100 different probes are methylated probes selected from the group consisting of SEQ. ID. NO. 14,974 through SEQ. ID. NO. 29,946.

26. The methylation detection array as defined in any one of claim 1 through claim 25, wherein the beads are silica beads.

27. The methylation detection array as defined in any one of claim 1 through claim 26, wherein each of the sample probes further includes a barcode sequence.

28. An array, comprising: at least 100 different probes, each of the at least 100 different probes including a respective sequence selected from the group consisting of SEQ. ID. NO. 1 through SEQ. ID. NO. 14,973 through SEQ. ID. NO. 29,946.

29. The array as defined in claim 28, wherein the array is a target capture panel and wherein the at least 100 different probes are to capture a respective target deoxyribonucleic acid strand.

30. The array as defined in claim 28, wherein the array is an enrichment panel and wherein the at least 100 different probes are to capture a respective target library fragment.31 . The array as defined in any one of claim 28 through claim 30, further comprising: a substrate having a plurality of depressions defined therein; and a plurality of beads, each of the plurality of beads positioned within one of the plurality of depressions; wherein each of the at least 100 different probes is attached to a respective one of the plurality of beads.

32. The array as defined in any one of claim 28 through claim 31 , wherein each of the at least 100 different probes further includes a barcode sequence.

33. A methylation detection kit, comprising: a methylation detection array, including: a substrate having a plurality of depressions defined therein; a plurality of beads, each of the plurality of beads positioned within one of the plurality of depressions; anda plurality of sample probes respectively attached to the plurality of beads, the plurality of sample probes including a plurality of probe sets, wherein: each of the plurality of probe sets includes an unmethylated probe and a corresponding methylated probe; and the unmethylated probe and the corresponding methylated probe are: i) each based on a bisulfite or enzymatically converted top strand sequence of a corresponding top strand sequence that contains a high minor allele frequency single nucleotide polymorphism where a reference is [G] followed by [CG]; or ii) each based on a bisulfite or enzymatically converted bottom strand sequence of a corresponding bottom strand sequence that contains a high minor allele frequency single nucleotide polymorphism where a reference is [G] followed by [N] and preceded by [CG]; or iii) each based on a bisulfite or enzymatically converted bottom strand sequence of a corresponding bottom strand sequence that contains a high minor allele frequency single nucleotide polymorphism where a reference is [C] followed by [G] and preceded by [CG]; and a sodium bisulfite solution; or an enzymatic methylation conversion mix.

34. A method comprising: generating a bisulfite converted or enzymatically converted and amplified DNA sample; and performing a methylation assay using the bisulfite converted or enzymatically converted and amplified DNA sample and the methylation detection array of claim 1.

35. The method as defined in claim 34, further comprising performing a genotyping reaction the bisulfite converted or enzymatically converted and amplified DNA sample at an exposed 3’ OH group.

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