Digital sequence analysis of DNA methylation

A method of characterizing a test sample comprising:

• i) providing a DNA detection assay that detects a methylation status of a set of at least three CpG loci in at least one marker DNA in said test sample, said DNA detection assay produced by a method comprising:
• selecting a set of at least three CpG loci by a process comprising:
• a) isolating marker DNA from a first plurality of known samples in which said marker DNA is unmethylated DNA that exhibits background methylation, and from a second plurality of known samples in which said marker DNA is methylated;
• b) treating said isolated marker DNA with bisulfite under conditions wherein unmethylated cytosine residues are converted to uracil residues to produce converted isolated marker DNA;
• c) processing said converted isolated marker DNA using digital sequencing and/or digital polymerase chain reaction (PCR) to determine the methylation status for three or more CpG loci in each of at least 1000 individual copies of said isolated marker DNA from each of said first plurality and said second plurality of known samples;
• d) for each of said three or more CpG loci, calculating a ratio between a mean methylation at that CpG locus in said first plurality of known samples to a mean methylation at the corresponding CpG locus in said second plurality of known samples;
• and
• e) selecting a defined set of at least three CpG loci for which the percentage of individual copies of the marker DNA from said first plurality of known samples that are methylated at all of said at least three CpG loci in said defined set is less than the percentage of individual copies of said marker DNA from said second plurality of known samples that are methylated at all of said at least three CpG loci in said defined set;
• and
• creating a DNA detection assay that detects the methylation status of all of the at least three CpG loci in said defined set of CpG loci in a strand of the bisulfite-treated marker DNA, said creating comprising synthesizing primer oligonucleotides that amplify a segment of bisulfite-treated marker DNA comprising said defined set of CpG loci;
• ii) isolating DNA from a test sample and treating said DNA from the test sample with bisulfite under conditions wherein unmethylated cytosine residues are converted to uracil residues to produce bi sulfite-treated marker DNA from said test sample, and
• iii) characterizing the methylation state of at least one marker DNA in said test sample, comprising detecting the methylation status of the set of at least three CpG loci in said bisulfite-treated marker DNA from said test sample using said DNA detection, wherein the absence of methylation at any of the CpG loci in said defined set of CpG loci in a strand of said bi sulfite-treated marker DNA from said test sample produces an assay result classifying that strand of marker DNA from the test sample as not methylated.