Digital sequence analysis of DNA methylation
The present invention relates to methods and compositions for determination of and uses of specific methylation patterns indicative of adenoma and carcinoma. In particular, the invention relates to analysis of defined CpG loci that are coordinately methylated in DNAs from cancer and adenoma samples, methods for identifying coordinately methylated loci, and methods of using analysis of coordinately methylated loci in one or more marker regions in the design of assays for adenoma and cancer.
1. A method of making an assay for detecting DNA amplified from a bisulfite-treated methylated marker DNA in the presence of DNA amplified from the bisulfite-treated unmethylated marker DNA that exhibits background methyation,
comprising:
a) isolating marker DNA from a first plurality of samples in which said marker DNA is unmethylated DNA exhibiting background methylation and from a second plurality of 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 a 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 samples;
d) for each of said three or more CpG loci, determining a ratio between a mean methylation at that CpG locus in said first plurality of samples to a mean methylation at the corresponding CpG locus in said second plurality of samples;
e) selecting a defined subset of at least three CpG loci for which the percentage of individual copies of the marker DNA from said first plurality of samples that are methylated at all of said at least three CpG loci in said defined subset is less than the percentage of individual copies of said marker DNA from said second plurality of samples that are methylated at all of said at least three CpG loci in said defined subset; and
f) creating a DNA detection assay that detects the methylation status of all of the at least three CpG loci in said defined subset of CpG loci in a strand of the bisulfite-treated marker DNA,
wherein said creating comprises synthesizing a primer oligonucleotides configured to amplify a segment of marker DNA comprising said defined subset of CpG loci, and wherein the absence of methylation at any of the CpG loci in said defined subset of CpG loci in a strand of said marker DNA produces an assay result classifying that strand of said marker DNA as not methylated.
2. The method of claim 1 , wherein the plurality of individual copies of a marker DNA analyzed in each of said first and said second plurality of samples comprises at least 10,000.
3. The method of claim 1 , wherein the plurality of individual copies of a marker DNA analyzed in each of said first and said second plurality of samples comprises at least 100,000.
4. The method of claim 1 , wherein each of said first and said second plurality of samples comprises at least 10.
5. The method of claim 1 , wherein each of said first and said second plurality of samples comprises at least 100.
6. The method of claim 1 , wherein said defined subset of CpG loci comprises at least four CpG loci.
7. method of claim 1 , wherein said defined subset of CpG loci comprises at least five CpG loci.
8. The method of claim 1 , wherein said DNA detection assay comprises at least one assay selected from the group consisting of a primer extension assay, a nucleic acid amplification assay, a structure-specific cleavage assay, 5′ nuclease cleavage assay, an invasive cleavage assay, and a ligation assay.