Methods for Quantification of Nucleosome Modifications and Mutations at Genomic Loci and Clinical Applications Thereof

A method for detecting and quantitating the presence of an epigenetic modification or a mutation at an epitope of a core element at a specific genomic locus in chromatin from a biological sample of a subject, the method comprising:

• a) isolating a biological sample from the subject;
• b) binding a cell, nucleus, organelle, or tissue comprising the core element having the epitope from the biological sample to a solid support;
• c) permeabilizing the cell, nucleus, organelle, or tissue;
• d) binding to a solid support a recombinant nucleosome standard comprising the core element having the epitope, the nucleosome standard comprising:
• a. a protein octamer, containing two copies each of histones H2A, H2B, H3, and H4, and optionally, linker histone H1;
• b. a DNA molecule, comprising:
• i. a nucleosome positioning sequence,
• ii. a DNA barcode;
• iii. a nuclease or transposase recognition sequence;
• and
• c. a binding member linked to the DNA molecule, wherein the binding member specifically binds to a binding partner;
• e) contacting the permeabilized cell, nucleus, organelle, or tissue of c) and the bound nucleosome standard of d) with an affinity reagent that specifically binds to the epitope;
• f) adding an affinity reagent-binding agent linked to a nuclease or transposase;
• g) allowing the nuclease or transposase to cleave DNA in the cell, nucleus, organelle, or tissue and the nuclease or transposase recognition sequence in the nucleosome standard;
• h) identifying the cleaved DNA;
• and
• i) detecting and quantitating the presence of the epitope at the genomic locus by comparing its abundance relative to the nucleosome standard.