Search

EP-4735629-A1 - METHYLATION DETECTION ARRAY AND KIT

EP4735629A1EP 4735629 A1EP4735629 A1EP 4735629A1EP-4735629-A1

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.

Inventors

  • BARNES, Bret D.
  • WESTPHAL, Maximillian S.

Assignees

  • Illumina, Inc.

Dates

Publication Date
20260506
Application Date
20240628

Claims (20)

  1. 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. 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. 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. 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. 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. 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. 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. 8. The methylation detection array as defined in one of claim 1 through claim 7, wherein the beads are silica beads.
  9. 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. 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, and G, 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. 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. 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. 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. 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. 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. 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. 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; and four different single probes.
  18. 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. 19. The methylation detection kit as defined in one of claim 14 through claim 18, wherein the plurality of sample probes is to capture respective target sequences from a mammal genome.
  20. 20. The methylation detection kit as defined in one of claim 14 through claim 19, further comprising sodium bisulfite solution.

Description

METHYLATION DETECTION ARRAY AND KIT CROSS-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 be designed 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, 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 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