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 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.
2. The method of claim 1 , wherein said defined set of CpG loci comprises at least four CpG loci.
3. The method of claim 1 , wherein said defined set of CpG loci comprises at least five CpG loci.
4. 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.
5. The method of claim 1 , wherein said test sample is a sample from a human.
6. The method of claim 5 , wherein said test sample from a human is a stool sample.
7. The method of claim 5 , wherein said at least one marker DNA comprises cancer and/or adenoma marker DNA.
8. The method of claim 7 , wherein said at least one marker DNA comprises at least three cancer or adenoma marker DNAs.
9. The method of claim 7 , wherein said at least one marker DNA comprises at least four cancer or adenoma marker DNAs.
10. The method of claim 7 , wherein said cancer or adenoma marker DNA is selected from the group consisting of vimentin, BMP3, Septin 9, TFPI2, 2 regions of LRAT, and EYA4 DNAs.
11. The method of claim 10 , wherein said at least one marker DNA comprises vimentin DNA, wherein said defined set of CpG loci in said vimentin DNA comprises loci 37, 40, and 45.
12. The method of claim 10 , wherein said at least one marker DNA comprises BMP3 DNA, wherein said defined set of CpG loci in said BMP3 DNA comprises loci 34, 53, and 61.
13. The method of claim 10 , wherein said at least one marker DNA comprises Septin9 DNA, wherein said defined set of CpG loci in said Septin9 DNA comprises loci 59, 61, 68, and 70.
14. The method of claim 10 , wherein said at least one marker DNA comprises TFPI2 DNA, wherein said defined set of CpG loci in said TFPI2 DNA comprises loci 55, 59, 63, and 67.
15. The method of claim 10 , wherein said at least one marker DNA comprises a EYA4 DNA, wherein said defined set of CpG loci in said EYA4 DNA comprises loci 31, 34, 37, and 44.
16. The method of claim 10 , wherein at least one marker DNA comprises vimentin, BMP3, Septin9, and TFPI2 DNA.