Methylation detection array and kit

Abstract

A methylation detection array includes 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; a plurality of sample probes respectively attached to some of the plurality of beads; and a plurality of lambda phage control probes respectively attached to some other of the plurality of beads. The plurality of lambda phage control probes includes a probe set including an unmethylated probe and a corresponding methylated probe; and a single probe. Each of the plurality of lambda phage control probes targets a cytosine in a CH site of a lambda phage target sequence and is free of additional CpG sites. The plurality of lambda phage control probes make up from greater than 0% to less than 5% of a total of the sample probes and the control probes.

Description

METHYLATION DETECTION ARRAY AND KITCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Serial Number 63 / 511 ,512, filed June 30, 2023, the content of which is incorporated by reference herein in its entirety.REFERENCE TO SEQUENCE LISTING

[0002] The Sequence Listing submitted herewith is hereby incorporated by reference in its entirety. The name of the file is ILI265BPCTJP-2650- PCT_Sequence_Listing.xml, the size of the file is 5,785,606 bytes, and the date of creation of the file is June 25, 2024.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 sample probes along with a small percentage of control probes that are based on the lambda phage genome. The sample probes are used to detect DNA methylation in a sample, and at least some of the control probes are used to detect methylation of a spike-in control. The control probes are sample independent, and thus will provide the same results regardless of the type of sample being tested. With the methylation array disclosed herein, the methylation assay is streamlined because control probes do not have to bedesigned for each individual sample. Moreover, the controls will enable comparisons to be made between runs of a sample on particular array or between different arrays.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 two example target strands, the strands after bisulfite conversion, and the strands after amplification, and probes that can be designed for the two example target strands;

[0009] Fig. 3 depicts an example of a target strand and the strand after enzymatic conversion; and

[0010] Fig. 4 is a schematic illustration of three depressions of the methylation array disclosed herein including three different control probes.DETAILED DESCRIPTION

[0011] 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, themethylation 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 found next to a guanine nucleotide (G), and where the C and G are linked by a phosphate group.

[0012] The methylation arrays disclosed herein also include control probes in addition to the sample probes. The control probes are based on the lambda phage genome, and are used to measure the quality of bisulfite or enzymatic conversion. During the assay, a lambda phage spike-in is added to the sample. This spike-in includes a known concentration of the lambda phage genome (referred to herein as the lambda phage target strands), which have been bisulfite or enzymatically converted and that are capable of hybridizing to respective bisulfite conversion control probes or enzymatic conversion control probes.

[0013] Both the bisulfite conversion and enzymatic conversion control probes disclosed herein target a CH locus (where “H” is A, T, or C) in the lambda phage genome, which should not be methylated. When single base extension is performed, the control probes and hybridized lambda phage target strands generate the same result over different runs and across different arrays. By targeting the C in the CH locus, the results from the bisulfite conversion control probe should not include a detected C, which would only be present after bisulfite conversion if a C in the original strand were methylated. With the ability to obtain consistent data from the bisulfite conversion control probes, a user can determine the quality of bisulfite conversion of the sample being analyzed. Similarly, by targeting the C in the CH locus, the results from the enzymatic conversion control probe should not include a detected A, which would only be present after enzymatic conversion if a C in the original strand were methylated. With the ability to obtain consistent data from the enzymatic conversion control probes, a user can determine the quality of enzymatic conversion of the sample being analyzed.

[0014] In addition to the bisulfite conversion or enzymatic conversion control probes, some examples of the methylation array also include specificity control probes. The specificity control probes are designed to monitor allele specific extension for the sample probes including two probes per CpG locus, and to monitorextension specificity for sample probes including one probe per CpG locus. In the examples disclosed herein, the specificity control probes are designed against nonpolymorphic A, T, C, or G sites of the lambda phage genome, and do not include underlying CpG sites. The specificity control probes should provide relatively consistent data, and thus can also be used to determine the quality of the bisulfite or enzymatic conversion.

[0015] In addition to the bisulfite or enzymatic conversion control probes and / or the specificity control probes, some examples of the methylation array also include nonpolymorphic control probes. The nonpolymorphic control probes are respectively designed to ensure that the array can bind and interpret each of the four nucleotides - A, T, C, and G. In the examples disclosed herein, the nonpolymorphic control probes enable a particular base - A, T, C, or G - in a nonpolymorphic region of the bisulfite or enzymatic converted lambda phage genome to be queried. With the nonpolymorphic controls, the signals for the hybridized, bisulfite converted lambda phage target should represent A (unmethylated), not C (methylated), and the signals for the hybridized, enzymatically converted lambda phage target should represent C (unmethylated), not A (methylated).

[0016] 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; a plurality of sample probes 18 respectively attached to some of the plurality of beads 16; and a plurality of lambda phage control probes 20 respectively attached to some other of the plurality of beads 16. As depicted in Fig. 1A, the methylation detection array 10 may also include a plurality of lambda phage specificity control probes 26 and / or a plurality of lambda phage nonpolymorphic control probes 28.

[0017] 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.

[0018] 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.

[0019] 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. In these 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., Ta2Os), aluminum oxide (e.g., AI2O3), silicon oxide (e.g., SiCh), 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.

[0020] 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.

[0021] 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.

[0022] 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 average pitch 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.

[0023] 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.

[0024] 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 depression14. 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.

[0025] 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 regular or 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.

[0026] In the examples disclosed herein, over 90% of the beads 16 in the array 10 have sample probes 18 attached thereto. In some examples, over 95% of the beads 16 in the array 10 have sample probes 18 attached thereto. In an example, each of the plurality of sample probes 18 is to capture respective target sequences from a mammal genome. As specific examples, the mammal genome may be the human genome or a dog genome.

[0027] The sample probes 18 can be categorized as two different types for each of the conversions described herein.

[0028] For bisulfite conversion, the first type includes two probes per CpG locus of the DNA sample strands. In this example, one of the two probes is for detectingthe unmethylated DNA state (i.e. , C converts to U converts to T) of a bisulfite converted DNA sample strand, and another of the probes is for detecting the methylated DNA state (i.e., C remains C) of the bisulfite converted DNA sample strand. The 3’ terminus of each probe is designed to match either the protected cytosine (methylated design) or the thymine base resulting from bisulfite conversion and whole-genome amplification (unmethylated design). Throughout this disclosure, sample and control probes specifically designed for detecting the unmethylated DNA state may be referred to as an unmethylated probe, and sample and control probes specifically designed for detecting the methylated DNA state may be referred to as a methylated probe.

[0029] With the first type of sample probes 18, when the bisulfite converted DNA target strands - with the unmethylated CpG sites - hybridize to the unmethylated probe, and when the bisulfite converted DNA target strands - with the methylated CpG sites - hybridize to the methylated probe, single-base extension is enabled. Singlebase 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.

[0030] For bisulfite conversion, the second type includes a single probe per CpG locus. The 3’ terminus of this probe 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.

[0031] For enzymatic conversion, the first type also includes two probes per CpG locus of the DNA sample strands. In this example, 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 DNAsample strand. The 3’ terminus of each probe is designed to match either the unmethylated cytosine or the thymine base resulting from enzymatic conversion.

[0032] Similar to the sample probes 18 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.

[0033] For enzymatic conversion, the second type includes a single probe per CpG locus. The 3’ terminus of this probe 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.

[0034] In an example, each of the sample probes 18 is a 50-mer, which is based on the assumption that methylation is regionally correlated within a 50 base pair (bp) span.

[0035] The sample probes 18 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 18 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. 2 illustrates one example of design principles that can be used for the sample probes 18 for bisulfite converted samples. In this example, two probes 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 a 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. 2. In contrast, a single probe (labeled “degenerate probe” in Fig. 2) is used fortargets having 4 or fewer CpG sites (the probe includes degenerate nucleotide R, complementary to II or C, at the C position of each CpG site). It is to be understood that other design principles may be used, such as those described in U.S. Patent No. 8,150,626, which is incorporated herein by reference in its entirety.

[0036] Similar design principles can be used for the sample probes 18 for enzymatically converted samples. An example of an enzymatically converted sample including 4 CpG sites is shown in Fig. 3. 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).

[0037] In the examples disclosed herein, from about 15% to about 20% of the sample probes 18 in the array 10 are of the first type, and from about 80% to about 85% of the sample probes 18 in the array 10 are of the second type.

[0038] An example of a commercially available array-based product including sample probes 18 (without the various control probes disclosed herein) for detection of methylation is the METHYLATIONEPIC™ BEADCHIP™ (from Illumina, Inc.) which allows interrogation of over 850,000 methylation sites quantitatively across the human genome at single-nucleotide resolution. The control probes 20, alone or in combination with the specificity control probes 26 and / or the nonpolymorphic control probes 28, disclosed herein may be incorporated into this type of array-based product.

[0039] Each array 10 includes a plurality of lambda phage control probes 20, shown as probes 20A, 20B, and 20C in Fig. 4. As shown in Fig. 1 , the array 10 may also include the specificity control probes 26 and / or the nonpolymorphic control probes 28. The control probes 20A, 20B, 20C, the specificity control probes 26, and the nonpolymorphic control probes 28 are designed to hybridize to corresponding segments of the lambda phage genome. The plurality of lambda phage control probes 20 specifically includes i) a probe set, which include an unmethylated probe 20A and acorresponding methylated probe 20B; and ii) a single probe 20C. The probes 20A and 20B of the set are similar to the first type of sample probes described herein, and the single probe 20C is similar to the second type of sample probe described herein. Similarly, the plurality of lambda phage specificity control probes 26 specifically includes i) a probe set, which include an unmethylated specificity probe and a corresponding methylated specificity probe; and ii) a single specificity probe; and the plurality of lambda phage nonpolymorphic control probes 28 specifically includes i) a probe set, which include an unmethylated nonpolymorphic probe and a corresponding methylated nonpolymorphic probe; and ii) a single nonpolymorphic probe.

[0040] The design principles for the plurality of lambda phage control probes 20 are i) that each probe 20 targets a cytosine in a CH site / locus of the lambda phage target sequence and ii) that each probe 20 is free of CpG sites. 122-mers of the lambda phage genome containing a CpG site are identified (see Table 1), and a CH site within the 122-mer is selected as the target. The control probes 20A, 20B, 20C are designed so that the CpG site is not included. The control probes 20A, 20B, 20C may be designed using the original sequence of the 122-mer, the bisulfite converted sequence, the complement of the bisulfite converted sequence, the enzymatically converted sequence, or the complement of the enzymatically converted sequence. By designing for CHs and prohibiting underlying CpG sites, the bisulfite conversion or enzymatic conversion can be directly measured using the probes 20A, 20B, 20C.

[0041] Table 1 depicts the 122-mer segments of the lambda phage genome used to design any of the probe sets (e.g., 20A, 20B) set forth in Table 2 and the single probes (20C) set forth in Table 3. The 122-mer segments may also be used to design the specificity probe sets (Table 4) or single probes (Table 5) and the nonpolymorphic probe sets (Table 6) or single probes (Table 7). In Table 1 , the chromosome and coordinate of each of the target sequences is identified, as well as the sequence identification number (SEQ. ID. NO.). Within each target sequence, the initially identified, but not included, CpG locus is identified by [CG]. The first section of Table 1 includes target sequences for which both a probe set and one or more single control probes have been designed, and the second section of Table 1 includes target sequence for which only one or more single control probes have been designed.TABLE 1

[0042] Each unmethylated probe 20A and its corresponding methylated probe 20B are based on the same lambda phage target sequence.

[0043] For the bisulfite conversion control probes 20A, 20B, the 3’ terminus of the unmethylated probe 20A is for detecting the unmethylated state of the bisulfite converted lambda phage sample strand, and the 3’ terminus of the methylated probe 20B is for detecting the methylated state of the bisulfite converted lambda phage sample strand. As mentioned, the CH locus of the control probes 20 should not lead to the detection of a C base. However, because cytosine methylation can occur outside of a CpG site, the methylated version of the probe 20B is also included. Examples of the unmethylated and corresponding methylated bisulfite conversion control probes 20A, 20B are identified in Table 2 by their sequence ID numbers. In Table 2, the unmethylated probe is designated as an odd numbered sequence in the group consisting of SEQ. ID. NO. 1795 through SEQ. ID. NO. 2735; and the corresponding methylated probe is designated as an even numbered sequence that isone number higher than the odd numbered sequence and is in the group consisting of SEQ. ID. NO. 1796 through SEQ. ID. NO. 2736. The sequence identification number (SEQ. ID. NO.) of the lambda phage target sequence used as the basis of the unmethylated and methylated probes 20A, 20B is also identified.

[0044] For the enzymatic conversion control probes 20A, 20B, the 3’ terminus of the unmethylated probe 20A is for detecting the unmethylated state of the enzymatically converted lambda phage sample strand, and the 3’ terminus of the methylated probe 20B is for detecting the methylated state of the enzymatically converted lambda phage sample strand. As mentioned, the CH locus of the control probes 20 should not lead to the detection of an A base. However, because cytosine methylation can occur outside of a CpG site, the methylated version of the probe 20B is also included. The probes shown in Table 2 can be used for enzymatically converted lambda phage sample strand, except that the corresponding methylated probe is designated as an odd numbered sequence in the group consisting of SEQ. ID. NO. 1795 through SEQ. ID. NO. 2735; and the unmethylated probe is designated as an even numbered sequence that is one number higher than the odd numbered sequence and is in the group consisting of SEQ. ID. NO. 1796 through SEQ. ID. NO. 2736.TABLE 2

[0045] Several single probes 20C are also disclosed herein. With the single probes 20C, single base extension results in the addition of a labeled G or A base, complementary to either the methylated C or T (indicative of an unmethylated C) of the bisulfite converted spike control (which, as described herein, should be unmethylated) or complementary to either the unmethylated C or T (indicative of a methylated C) of the enzymatically converted spike control (which, as described herein, should be unmethylated).

[0046] As shown in Table 3, in some examples, one single probe 20C has been identified for one lambda phage target sequence, and in other examples, multiple single probes 20C have been identified for one lambda phage target sequence. For the control probes 20C, the sequence identification number (SEQ. ID. NO.) of the lambda phage target sequence used as the basis of the single probes 20C is also identified. In Table 3, the single probe 10C is designated as one of SEQ. ID. NO. 2737 through SEQ. ID. NO. 4648.Table 3

[0047] Each of the control probes 20 (e.g., 20A, 20B, 20C) is a 50-mer.

[0048] Fig. 4 depicts a portion of the array 10, where each depression 14 includes a bead 16 with one of the control probes 20A, 20B, 20C respectively attached thereto. In one example, the plurality of lambda phage control probes (i.e. , the total of 20A through 20C) make up from greater than 0% to less than 5% of a total of thesample probes 18 and the control probes 20. As one example, the plurality of lambda phage control probes 20 makes up from about 1 % to about 3% of the total of the sample probes 18 and the control probes 20. The plurality of lambda phage control probes 20 may include from 5 to 100 different probe sets (5-100 of each of 20A and its corresponding 20B); and from 4 to 100 different single probes (4-100 of each of 20C). In one specific example, the total number of probes 18+20 is 360, and the plurality of lambda phage control probes 20 includes five (5) different probe sets (5 of each of 20A and its corresponding 20B); and four (4) different single probes (4 of each of 20C). Any of the probe sets of Table 2 and any of the probe sets of Table 3 may be used.

[0049] The design principles for the plurality of lambda phage specificity control probes 26 are i) that each probe 26 targets a cytosine in a CH or CG site / locus of the lambda phage target sequence and ii) that each probe 26 is free of CpG sites. The 122-mers of the lambda phage genome containing a CpG site that are identified in Table 1 may be used, and a CH or CG site within the 122-mer is selected as the target. The specificity control probes 26 are designed so that the CpG site is not included. The specificity control probes 26 may be designed using the original sequence of the 122-mer, the bisulfite converted sequence, the complement of the bisulfite converted sequence, the enzymatically converted sequence, or the complement of the enzymatically converted sequence.

[0050] The specificity control probes 26 can be designed to monitor allelespecific extension for the sample probes 18 of the first type (i.e. , two probes). The methylation status of a particular cytosine (of the CH or CG site) is carried out following bisulfite or enzymatic treatment of DNA by using query probes for the unmethylated and methylated state of each CpG locus. Similar to the control probes 20 for bisulfite conversion, the A / T match corresponds to the unmethylated status of the interrogated C, and the G / C match corresponds to the methylated status of C. Similar to the control probes 20 for enzymatic conversion, the A / T match corresponds to the methylated status of the interrogated C, and the G / C match corresponds to the unmethylated status of C. The matched pairs enable the extension reaction with labeled nucleotides, which are subsequently stained with a fluorescent reagent. A G / Tmismatch prevents extension, and thus should result in no or low signals. Examples of these probe sets are identified in Table 4.TABLE 4

[0051] The specificity control probes 26 can also monitor extension specificity for the second type of sample probe 18 disclosed herein. For bisulfite converted lambda phage target sequences, these specificity control probes 26 incorporate the A base across a nonpolymorphic T and have intensity in the red channel. If there is nonspecific incorporation of the G base, the probe has elevated signal in the greenchannel. These probes can also be designed for enzymatic conversion, where the G base is incorporated across a nonpolymorphic C and has intensity in the green channel. If there is nonspecific incorporation of the A base, the probe has elevated signal in the red channel. Examples of these probe sets are identified in Table 5.TABLE 5

[0052] The design principle for the plurality of lambda phage nonpolymorphic control probes 28 is that the 3’ terminus of each probe is designed to interrogate one of the four bases - A, T, C, and G - in a nonpolymorphic region of the bisulfite converted or enzymatically converted lambda phage sample. For the bisulfite converted lambda phage sample, it is desirable for the A bases to be incorporated and read, and for the enzymatic converted lambda phage sample, it is desirable for the G bases to be incorporated and read. Examples of two nonpolymorphic control probes 28 are shown in Table 6 and examples of single nonpolymorphic control probes 28 are shown in Table 7.TABLE 6TABLE 7

[0053] Each of the probes 18, 20 may include a decoder portion at its 5’ end, shown at reference numerals 22A through 22C in Fig. 3). The decoder portion is a nucleotide sequence that may be used to distinguish individual beads 16. The decoder 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.

[0054] In the examples disclosed herein, all of the probes 18 and 20 are attached to respective bead 16. While a single sample probe 18 and a single control probe 20 are 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 (e.g., at the end of the decoder portion 22A, 22B, 22C) 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.

[0055] 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 biontinylated probe 18, 20.

[0056] 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.

[0057] The array 10 may be included in a methylation detection kit. This kit includes any example of the array 10 disclosed herein. In some instances, the kit also includes a lambda phage spike-in solution, including a liquid carrier and a predetermined concentration of the lambda phage genome (lambda phage DNA target strands). In other instances, the lambda phage spike-in solution may be obtained separately from the array 10 (e.g., commercially available from New England BioLabs Inc.). The liquid carrier of the lambda phage spike-in solution may be water and / or an ionic salt buffer fluid, e.g., saline citrate at milli-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 lambda phage target strands includeSEQ. ID. NO. 1 through SEQ. ID. NO. 1794; and the plurality of lambda phage control probes 20A and 20B or 20C correspond with at least one of the lambda phage target strands.

[0058] In the example array 10 including five different control probe sets (5 of each of 20A and its corresponding 20B) and four different single control probes (4 of each of 20C), the lambda phage spike-in solution includes lambda phage target strands of SEQ. ID. NO. 1 through SEQ. ID. NO. 1714, where one of SEQ. ID. NO. 1 through SEQ. ID. NO. 1714 corresponds with at least one of the five different probe sets, at least one of the four different single probes, or at least one of the five different probe sets and at least one of the four different single probes. The array may also include the control specificity probes 26 and / or the control nonpolymorphic probes 28 disclosed herein.

[0059] The kit may further include a sodium bisulfite solution that can be used in the bisulfite conversion of the DNA sample that is to be used with the array 10.

[0060] One example of the method for using the array 10 includes generating a bisulfite converted and amplified DNA sample; spiking the bisulfite converted and amplified DNA sample with the lambda phage spike-in solution disclosed herein, thereby generating a spiked sample; and performing a methylation assay using the spiked sample and the methylation detection array 10.

[0061] For the bisulfite conversion, DNA is first denatured (made singlestranded) and then treated with sodium bisulfite. The converted DNA is also amplified. A predetermined amount of the lambda phage spike-in solution may be added, which is based upon the percentage of control probes 20 on the array 10. In one example, a minimum of 1 % of the spike-in solution is added. The methylation assay enables hybridization of the converted and amplified DNA sample strands and the lambda phage target strands to the sample probes 18 and control probes 20, respectively.

[0062] The kit may alternatively include 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.

[0063] The enzymatic methylation conversion mix includes a liquid carrier and an altered cytidine deaminase. Examples of this mix are described in InternationalPatent 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.

[0064] 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.

[0065] The enzymatic methylation conversion also includes the altered cytidine deaminase.

[0066] 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, the altered 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. 5275). 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. 5276, whereinX 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. 5277, 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. 5276 or SEQ. ID. NO. 5277, 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. 5278). Specific examples of altered cytidine deaminases having increased activity and preferentially acting on 5mC compared to cytosine include SEQ. ID. NO. 5278 or a sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity to SEQ. ID. NO. 5278 and including Y130A.

[0067] 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. 5275). 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 positively charged 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.: 5275, 5279, or 5278 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. 5280 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. 5281 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.

[0068] 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 ^M. As examples instances, the concentration of the enzyme can be at least 0.5 ^M, or at least 1 ^M, or at least 2 ^iM, or at least 3 ^M, or at least 4 |iM, or 5 ^M, and / or no greater than 5 ^M, or no greater than 4 ^M, or no greater than 3 JJ.M, or no greater than 2 ^M, or no greater than 1 M. In some specific examples, the concentration of the enzyme can be about 0.4 |iM, or about 0.5 ^M, or about 0.8 ^M.

[0069] Another example of the method for using the array 10 includes generating an enzymatically converted DNA sample; amplifying enzymatically converted DNA sample; spiking the enzymatically converted and amplified DNA sample with the lambda phage spike-in solution disclosed herein, thereby generating a spiked sample; and performing a methylation assay using the spiked sample and the methylation detection array 10.

[0070] For the enzymatic conversion, DNA is first denatured (made singlestranded) and then treated with the enzymatic methylation conversion mix. During exposure of the DNA to the enzymatic methylation conversion mix, the conversion of5-methylcytosine (5mC) to thymidine (T) by deamination takes place at a greater rate than conversion of cytosine (C) to uracil (U) by deamination, resulting in a converted single-stranded DNA fragment (see the example shown in Fig. 3). The converted DNA is also amplified. A predetermined amount of the lambda phage spike-in solution may be added, which is based upon the percentage of control probes 20 on the array 10. In one example, a minimum of 1 % of the spike-in solution is added. The methylation assay enables hybridization of the converted and amplified DNA sample strands and the lambda phage target strands to the sample probes 18 and control probes 20, respectively. When included some of the lambda phage target strands also hybridize to the specificity control probes 26 and / or the nonpolymorphic control probes 28.

[0071] Regardless of the type of conversion that is formed, an extension reaction is performed (or at least attempted) at the 3’ ends of the probes 18, 20 (e.g., 20A, 20B, 20C), 26, 28. 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 and thymine. 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 or 20 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 |3, 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 milli-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 may also include catalytic metal(s) intended for the extension reaction, such as Mg2+, Mn2+, etc. A singlecatalytic 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.

[0072] 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, 26, 28. As described, the extension reaction (or lack thereof) enables one to determine the methylation status.

[0073] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein. It should also be appreciated that terminology explicitly employed herein that also may appear in any disclosure incorporated by reference should be accorded a meaning most consistent with the particular concepts disclosed herein.

[0074] The singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[0075] The terms comprising, including, containing and various forms of these terms are synonymous with each other and are meant to be equally broad.

[0076] 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).

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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; a plurality of sample probes respectively attached to some of the plurality of beads; and a plurality of lambda phage control probes respectively attached to some other of the plurality of beads, wherein: the plurality of lambda phage control probes includes: a probe set including an unmethylated probe and a corresponding methylated probe; and a single probe; each of the plurality of lambda phage control probes targets a cytosine in a CH site of a lambda phage target sequence and is free of additional CpG sites; and the plurality of lambda phage control probes make up from greater than 0% to less than 5% of a total of the sample probes and the control probes.

2. The methylation detection array as defined in claim 1 , wherein: the plurality of lambda phage control probes is for a bisulfite converted lambda phage target sequence; the unmethylated probe is designated as an odd numbered sequence in the group consisting of SEQ. ID. NO. 1795 through SEQ. ID. NO. 2735; the corresponding methylated probe is designated as an even numbered sequence that is one number higher than the odd numbered sequence and is in the group consisting of SEQ. ID. NO. 1796 through SEQ. ID. NO. 2736; and the single probe designated as one of SEQ. ID. NO. 2737 through SEQ. ID.NO. 4648.

3. The methylation detection array as defined in claim 1 or claim 2, wherein the plurality of lambda phage control probes includes: five different probe sets; and four different single probes.

4. The methylation detection array as defined in one of claim 1 through claim 3, wherein each of the plurality of sample probes is to capture respective target sequences from a mammal genome.

5. The methylation detection array as defined in one of claim 1 through claim 4, wherein each of the plurality of sample probes is to capture respective target sequences from a human genome.

6. The methylation detection array as defined in claim 1 , wherein each of the plurality of lambda phage control probes further includes a decoder sequence at its 5’ end.

7. The methylation detection array as defined in one of claim 1 through claim 6, wherein the plurality of lambda phage control probes make up from about 1% to about 3% of the total of the sample probes and the control probes.

8. The methylation detection array as defined in one of claim 1 through claim 7, wherein the beads are silica beads.

9. The methylation detection array as defined in one of claim 1 through claim 8, further comprising a plurality of lambda phage specificity control probes respectively attached to still some other of the plurality of beads, wherein: the plurality of lambda phage specificity control probes includes: a specificity probe set including:an unmethylated specificity probe selected from the group consisting of SEQ. ID. NO. 4649 through SEQ. ID. NO. 4692 and SEQ. ID. NO. 5282; and a corresponding methylated specificity probe selected from the group consisting of SEQ. ID. NO. 4693 through SEQ. ID. NO. 4737; and a single specificity probe designated as one of SEQ. ID. NO. 4738 through SEQ. ID. NO. 4768.

10. The methylation detection array as defined in one of claim 1 through claim 9, further comprising a plurality of lambda phage nonpolymorphic control probes respectively attached to still some other of the plurality of beads, wherein: the plurality of lambda phage nonpolymorphic control probes includes: a respective nonpolymorphic probe set targeted for A, T, C, andG, each of the respective nonpolymorphic probe sets including an unmethylated nonpolymorphic probe and a methylated nonpolymorphic probe; and a respective single nonpolymorphic probe targeted for A, T, C, and G.

11. The methylation detection array as defined in claim 10, wherein: the respective nonpolymorphic probe set targeted for A includes: an unmethylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 4769 through SEQ. ID. NO. 4783 and a corresponding methylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 4955 through SEQ. ID. NO. 4969; or an unmethylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 4821 through SEQ. ID. NO. 4856 and a corresponding methylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 5007 through SEQ. ID. NO. 5042; the respective nonpolymorphic probe set targeted for T includes:an unmethylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 4784 through SEQ. ID. NO. 4802 and a corresponding methylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 4970 through SEQ. ID. NO. 4988; or an unmethylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 4857 through SEQ. ID. NO. 4918 and a corresponding methylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 5043 through SEQ. ID. NO. 5104; the respective nonpolymorphic probe set targeted for C includes: an unmethylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 4803 through SEQ. ID. NO. 4814 and a corresponding methylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 4989 through SEQ. ID. NO. 5000; or an unmethylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 4919 through SEQ. ID. NO. 4926 and a corresponding methylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 5105 through SEQ. ID. NO. 5112; and the respective nonpolymorphic probe set targeted for G includes: an unmethylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 4815 through SEQ. ID. NO. 4820 and a corresponding methylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 5001 through SEQ. ID. NO. 5006; or an unmethylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 4927 through SEQ. ID. NO. 4954 and a corresponding methylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 5113 through SEQ. ID. NO. 5140.

12. The methylation detection array as defined in one of claim 10 or claim 11 , wherein: the respective single nonpolymorphic probe targeted for A is selected from the group consisting of SEQ. ID. NO. 5141 through SEQ. ID. NO. 5176;the respective single nonpolymorphic probe targeted for T is selected from the group consisting of SEQ. ID. NO. 5177 through SEQ. ID. NO. 5238; the respective single nonpolymorphic probe targeted for C is selected from the group consisting of SEQ. ID. NO. 5239 through SEQ. ID. NO. 5246; and the respective single nonpolymorphic probe targeted for G is selected from the group consisting of SEQ. ID. NO. 5247 through SEQ. ID. NO. 5274.

13. The methylation detection array as defined in claim 1 , wherein: the plurality of lambda phage control probes is for an enzymatically converted lambda phage target sequence; the corresponding methylated probe is designated as an odd numbered sequence in the group consisting of SEQ. ID. NO. 1795 through SEQ. ID. NO. 2735; the unmethylated probe is designated as an even numbered sequence that is one number higher than the odd numbered sequence and is in the group consisting of SEQ. ID. NO. 1796 through SEQ. ID. NO. 2736; and the single probe designated as one of SEQ. ID. NO. 2737 through SEQ. ID. NO. 4649 and SEQ. ID. NO. 5282.

14. 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; a plurality of sample probes respectively attached to some of the plurality of beads; and a plurality of lambda phage control probes respectively attached to some other of the plurality of beads, wherein: the plurality of lambda phage control probes includes: a probe set including an unmethylated probe and a corresponding methylated probe; and a single probe;each of the plurality of lambda phage control probes targets a cytosine in a CH site of a probe lambda phage target sequence and is free of additional CpG sites; and the plurality of lambda phage control probes make up from greater than 0% to less than 5% of a total of the sample probes and the control probes; and a lambda phage spike-in solution, including: a liquid carrier; and a predetermined concentration of lambda phage target strands.

15. The methylation detection kit as defined in claim 14, wherein: the lambda phage target strands include SEQ. ID. NO. 1 through SEQ. ID. NO. 1714; and the plurality of lambda phage control probes corresponds with at least one of the lambda phage target strands.

16. The methylation detection kit as defined in one of claim 14 or claim 15, wherein: the plurality of lambda phage control probes is for a bisulfite converted lambda phage target sequence; the unmethylated probe is designated as an odd numbered sequence in the group consisting of SEQ. ID. NO. 1795 through SEQ. ID. NO. 2735; the corresponding methylated probe is designated as an even numbered sequence that is one number higher than the odd numbered sequence and is in the group consisting of SEQ. ID. NO. 1796 through SEQ. ID. NO. 2736; and the single probe designated as one of SEQ. ID. NO. 2737 through SEQ. ID. NO. 4648.

17. The methylation detection kit as defined in one of claim 14 or claim 15, wherein the plurality of lambda phage control probes includes: five different probe sets; andfour different single probes.

18. The methylation detection kit as defined in claim 17, wherein: the lambda phage target strands include SEQ. ID. NO. 1 through SEQ. ID. NO. 1714; and one of SEQ. ID. NO. 1 through SEQ. ID. NO. 1714 corresponds with at least one of the five different probe sets, at least one of the four different single probes, or at least one of the five different probe sets and at least one of the four different single probes.

19. The methylation detection kit as defined in one of claim 14 through claim18, wherein the plurality of sample probes is to capture respective target sequences from a mammal genome.

20. The methylation detection kit as defined in one of claim 14 through claim19, further comprising sodium bisulfite solution.

21. The methylation detection kit as defined in one of claim 14, claim 15, or claim 19, further comprising an enzymatic methylation conversion mix.

22. The methylation detection kit as defined in one of claim 14 through claim 21 , wherein each of the plurality of lambda phage control probes further includes a decoder sequence at its 5’ end.

23. The methylation detection kit as defined in one of claim 14 through claim 21 , wherein the plurality of lambda phage control probes make up from about 1 % to about 3% of the total of the sample probes and the control probes.

24. The methylation detection kit as defined in one of claim 14 through claim 23, further comprising a plurality of lambda phage specificity control probes respectively attached to still some other of the plurality of beads, wherein:the plurality of lambda phage specificity control probes includes: a specificity probe set including: an unmethylated specificity probe selected from the group consisting of SEQ. ID. NO. 4649 through SEQ. ID. NO. 4692 and SEQ. ID. NO. 5282; and a corresponding methylated specificity probe selected from the group consisting of SEQ. ID. NO. 4693 through SEQ. ID. NO. 4737; and a single specificity probe designated as one of SEQ. ID. NO. 4738 through SEQ. ID. NO. 4768.

25. The methylation detection array as defined in one of claim 14 through claim 24, further comprising a plurality of lambda phage nonpolymorphic control probes respectively attached to still some other of the plurality of beads, wherein: the plurality of lambda phage nonpolymorphic control probes includes: a respective nonpolymorphic probe set targeted for A, T, C, andG, each of the respective nonpolymorphic probe sets including a unmethylated nonpolymorphic probe and a methylated nonpolymorphic probe; and a respective single nonpolymorphic probe targeted for A, T, C, and G.

26. The methylation detection kit as defined in claim 25, wherein: the respective nonpolymorphic probe set targeted for A includes: an unmethylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 4769 through SEQ. ID. NO. 4783 and a corresponding methylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 4955 through SEQ. ID. NO. 4969; or an unmethylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 4821 through SEQ. ID. NO. 4856 and acorresponding methylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 5007 through SEQ. ID. NO. 5042; the respective nonpolymorphic probe set targeted for T includes: an unmethylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 4784 through SEQ. ID. NO. 4802 and a corresponding methylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 4970 through SEQ. ID. NO. 4988; or an unmethylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 4857 through SEQ. ID. NO. 4918 and a corresponding methylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 5043 through SEQ. ID. NO. 5104; the respective nonpolymorphic probe set targeted for C includes: an unmethylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 4803 through SEQ. ID. NO. 4814 and a corresponding methylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 4989 through SEQ. ID. NO. 5000; or an unmethylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 4919 through SEQ. ID. NO. 4926 and a corresponding methylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 5105 through SEQ. ID. NO. 5112; and the respective nonpolymorphic probe set targeted for G includes: an unmethylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 4815 through SEQ. ID. NO. 4820 and a corresponding methylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 5001 through SEQ. ID. NO. 5006; or an unmethylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 4927 through SEQ. ID. NO. 4954 and a corresponding methylated nonpolymorphic probe selected from the group consisting of SEQ. ID. NO. 5113 through SEQ. ID. NO. 5140.

27. The methylation detection kit as defined in one of claim 25 or claim 26, wherein: the respective single nonpolymorphic probe targeted for A is selected from the group consisting of SEQ. ID. NO. 5141 through SEQ. ID. NO. 5176; the respective single nonpolymorphic probe targeted for T is selected from the group consisting of SEQ. ID. NO. 5177 through SEQ. ID. NO. 5238; the respective single nonpolymorphic probe targeted for C is selected from the group consisting of SEQ. ID. NO. 5239 through SEQ. ID. NO. 5246; and the respective single nonpolymorphic probe targeted for G is selected from the group consisting of SEQ. ID. NO. 5247 through SEQ. ID. NO. 5274.

28. A method comprising: generating a bisulfite converted or enzymatically converted and amplified DNA sample; spiking the bisulfite converted or enzymatically converted and amplified DNA sample with a lambda phage spike-in solution, including: a liquid carrier; and a predetermined concentration of lambda phage target strands, thereby generating a spiked sample; and performing a methylation assay using the spiked sample and the methylation detection array of one of claim 1 through claim 13.

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