Digital sequence analysis of DNA methylation

1. A method of making an assay for detecting DNA amplified from a methylated marker DNA in the presence of DNA amplified from the unmethylated marker DNA that exhibits background methylation, the method comprising:

a) isolating marker DNA from a first group of samples in which the marker DNA is unmethylated DNA exhibiting background methylation and from a second group of samples in which the marker DNA is methylated;

b) processing the 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 the isolated marker DNA from each of the first and second groups of samples;

c) for each of the three or more CpG loci, determining a ratio between a mean methylation at that CpG locus in the first group of samples to a mean methylation at the corresponding CpG locus in the second group of samples;

d) selecting a subset of at least three CpG loci for which the percentage of individual copies of the marker DNA from the first group of samples that are methylated at all of the at least three CpG loci in the subset is less than the percentage of individual copies of the marker DNA from the second group of samples that are methylated at all of the at least three CpG loci in the subset; and

e) creating a detection assay that detects the methylation status of all of the at least three CpG loci in the subset of CpG loci in a strand of the marker DNA, wherein the absence of methylation at any one of the CpG loci in the subset of CpG loci in a strand of the marker DNA produces an assay result classifying that strand of the marker DNA as not methylated,

wherein creating the detection assay comprises synthesizing at least one oligonucleotide complementary to a segment of nucleic acid comprising the subset of CpG loci.

2. The method of claim 1 , wherein the methylation status for the at least three CpG loci is determined in each of at least 10,000 individual copies of the isolated marker DNA from each of the first and second groups of samples.

3. The method of claim 1 , wherein the methylation status for the at least three CpG loci is determined in each of at least 100,000 individual copies of the isolated marker DNA from each of the first and second groups of samples.

4. The method of claim 1 , wherein each of the first and second groups of samples comprises at least 10 samples.

5. The method of claim 1 , wherein each of the first and second groups of samples comprises at least 100 samples.

6. The method of claim 1 , wherein the subset of CpG loci comprises at least four CpG loci.

7. The method of claim 1 , wherein the subset of CpG loci comprises at least five CpG loci.

8. The method of claim 1 , wherein the 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.

9. A method of characterizing a test sample comprising:

i) providing a detection assay for at least one marker DNA created according to claim 1 ;

ii) isolating strands of each of the at least one marker DNA from a test sample; and

iii) characterizing a set of at least three CpG loci in the strands of each of the at least one marker DNA from the test sample using the detection assay to produce assay results classifying strands of each of the at least one marker DNA from the test sample as methylated or not methylated.

10. The method of claim 9 , wherein the set of CpG loci comprises at least four CpG loci.

11. The method of claim 9 , wherein the set of CpG loci comprises at least five CpG loci.

12. The method of claim 9 , wherein the test sample is a sample from a human.

13. The method of claim 12 , wherein the test sample from a human is a stool sample.

14. The method of claim 9 , wherein the at least one marker DNA comprises cancer and/or adenoma marker DNA.

15. The method of claim 9 , wherein the at least one marker DNA comprises at least three cancer or adenoma marker DNAs.

16. The method of claim 9 , wherein the at least one marker DNA comprises at least four cancer or adenoma marker DNAs.

17. The method of claim 9 , wherein the at least one marker DNA is selected from the group consisting of the marker DNAs NM_000038, NM_000044, AB033043, AK055404, AK090480, BC041476, BX648962, NM_000017, NM_000022, NM_000038, NM_000038, NM_000043, NM_000044, NM_000044, NM_000076, NM_000077, NM_000077, NM_000077, NM_000088, NM_000095, NM_000104, NM_000115, NM_000115, NM_000115, NM_000125, NM_000125, NM_000125, NM_000125, NM_000182, NM_000193, NM_000249, NM_000249, NM_000280, NM_000280, NM_000280, NM_000308, NM_000314, NM_000321, NM_000321, NM_000336, NM_000362, NM_000362, NM_000362, NM_000378, NM_000402, NM_000438, NM_000443, NM_000453, NM_000453, NM_000475, NM_000492, NM_000492, NM_000514, NM_000517, NM_000520, NM_000524, NM_000551, NM_000551, NM_000551, NM_000610, NM_000610, NM_000612, NM_000620, NM_000680, NM_000717, NM_000721, NM_000782, NM_000799, NM_000813, NM_000818, NM_000829, NM_000830, NM_000834, NM_000843, NM_000852, NM_000852, NM_000852, NM_000857, NM_000863, NM_000902, NM_000914, NM_000915, NM_000926, NM_000927, NM_000959, NM_000965, NM_000965, NM_000965, NM_000997, NM_001001336, NM_001001723, NM_001002295, NM_001003689, NM_001003891, NM_001007792, NM_001008503, NM_001008504, NM_001008505, NM_001009598, NM_001011545, NM_001012331, NM_001013464, NM_001018084, NM_001020658, NM_001024844, NM_001025205, NM_001025604, NM_001033044, NM_001033056, NM_001033518, NM_001033519, NM_001033520, NM_001033952, NM_001036, NM_001043, NM_001053, NM_001059, NM_001063, NM_001100, NM_001109, NM_001176, NM_001186, NM_001204, NM_001228, NM_001250, NM_001257, NM_001319, NM_001325, NM_001385, NM_001451, NM_001454, NM_001458, NM_001480, NM_001538, NM_001553, NM_001572, NM_001628, NM_001635, NM_001651, NM_001718, NM_001753, NM_001753, NM_001768, NM_001801, NM_001851, NM_001883, NM_001884, NM_001927, NM_001954, NM_001958, NM_001972, NM_001975, NM_001989, NM_002007, NM_002012, NM_002024, NM_002065, NM_002110, NM_002127, NM_002148, NM_002155, NM_002191, NM_002212, NM_002221, NM_002235, NM_002253, NM_002344, NM_002412, NM_002457, NM_002478, NM_002529, NM_002588, NM_002658, NM_002700, NM_002807, NM_002848, NM_002873, NM_002923, NM_003027, NM_003088, NM_003097, NM_003149, NM_003204, NM_003219, NM_003238, NM_003238, NM_003246, NM_003274, NM_003277, NM_003295, NM_003300, NM_003391, NM_003392, NM_003408, NM_003417, NM_003426, NM_003435, NM_003451, NM_003474, NM_003508, NM_003539, NM_003540, NM_003541, NM_003546, NM_003666, NM_003735, NM_003736, NM_003775, NM_003777, NM_003806, NM_003823, NM_003888, NM_003914, NM_003923, NM_003984, NM_003991, NM_003999, NM_004004, NM_004064, NM_004068, NM_004102, NM_004113, NM_004122, NM_004135, NM_004181, NM_004230, NM_004248, NM_004267, NM_004291, NM_004297, NM_004327, NM_004360, NM_004360, NM_004360, NM_004378, NM_004385, NM_004387, NM_004394, NM_004411, NM_004441, NM_004464, NM_004477, NM_004480, NM_004484, NM_004525, NM_004530, NM_004612, NM_004621, NM_004714, NM_004737, NM_004787, NM_004817, NM_004865, NM_004887, NM_004929, NM_004936, NM_004936, NM_004938, NM_004975, NM_004976, NM_004988, NM_005032, NM_005048, NM_005073, NM_005100, NM_005117, NM_005117, NM_005157, NM_005159, NM_005181, NM_005233, NM_005284, NM_005285, NM_005286, NM_005346, NM_005382, NM_005386, NM_005398, NM_005427, NM_005437, NM_005523, NM_005556, NM_005584, NM_005638, NM_005668, NM_005806, NM_005825, NM_005946, NM_005959, NM_006000, NM_006019, NM_006041, NM_006043, NM_006053, NM_006142, NM_006158, NM_006158, NM_006161, NM_006194, NM_006211, NM_006306, NM_006307, NM_006463, NM_006483, NM_006484, NM_006497, NM_006497, NM_006539, NM_006587, NM_006614, NM_006735, NM_006765, NM_006788, NM_006874, NM_006898, NM_006917, NM_007117, NM_007181, NM_007182, NM_007182, NM_007182, NM_007197, NM_007294, NM_007294, NM_007332, NM_007345, NM_007361, NM_012200, NM_012202, NM_012261, NM_012295, NM_012295, NM_012301, NM_012309, NM_012309, NM_012399, NM_012444, NM_012458, NM_013250, NM_013291, NM_013381, NM_013435, NM_013942, NM_014020, NM_014080, NM_014155, NM_014228, NM_014234, NM_014325, NM_014379, NM_014386, NM_014459, NM_014468, NM_014522, NM_014574, NM_014587, NM_014588, NM_014618, NM_014618, NM_014631, NM_014653, NM_014676, NM_014710, NM_014724, NM_014786, NM_014817, NM_014979, NM_015002, NM_015094, NM_015101, NM_015163, NM_015472, NM_015507, NM_015610, NM_015641, NM_015683, NM_015722, NM_015920, NM_016003, NM_016135, NM_016157, NM_016162, NM_016179, NM_016192, NM_016192, NM_016223, NM_016229, NM_016301, NM_016442, NM_016521, NM_016535, NM_016538, NM_016540, NM_016552, NM_016568, NM_016605, NM_016931, NM_016950, NM_016954, NM_017514, NM_017649, NM_017729, NM_017798, NM_017844, NM_017847, NM_018061, NM_018074, NM_018129, NM_018135, NM_018197, NM_018310, NM_018354, NM_018401, NM_018431, NM_018898, NM_018899, NM_018901, NM_018906, NM_018911, NM_018920, NM_018925, NM_018926, NM_018927, NM_018928, NM_018950, NM_018976, NM_018997, NM_019043, NM_019102, NM_020166, NM_020201, NM_020208, NM_020226, NM_020230, NM_020348, NM_020469, NM_020549, NM_020630, NM_020650, NM_020657, NM_020660, NM_020685, NM_020815, NM_020873, NM_020984, NM_020985, NM_020986, NM_020999, NM_021032, NM_021101, NM_021179, NM_021193, NM_021216, NM_021257, NM_021614, NM_021911, NM_021926, NM_021956, NM_022076, NM_022088, NM_022169, NM_022405, NM_022443, NM_022468, NM_022469, NM_022718, NM_022750, NM_023926, NM_024012, NM_024046, NM_024101, NM_024306, NM_024409, NM_024504, NM_024593, NM_024600, NM_024826, NM_024882, NM_024893, NM_024944, NM_025019, NM_025058, NM_025061, NM_025087, NM_025197, NM_025204, NM_025208, NM_025218, NM_025263, NM_030577, NM_030667, NM_030668, NM_030669, NM_030670, NM_030671, NM_030760, NM_030806, NM_030920, NM_031277, NM_031283, NM_031424, NM_031466, NM_031488, NM_031497, NM_031856, NM_031859, NM_031860, NM_031882, NM_031883, NM_031901, NM_031912, NM_031922, NM_031934, NM_031994, NM_032034, NM_032087, NM_032094, NM_032098, NM_032099, NM_032100, NM_032101, NM_032109, NM_032134, NM_032140, NM_032192, NM_032256, NM_032303, NM_032391, NM_032402, NM_032403, NM_032406, NM_032411, NM_032412, NM_032603, NM_032625, NM_032803, NM_032825, NM_032838, NM_032883, NM_032918, NM_032945, NM_032961, NM_032967, NM_032968, NM_032969, NM_033126, NM_033135, NM_033143, NM_033224, NM_033302, NM_033303, NM_033304, NM_033445, NM_033624, NM_052902, NM_052954, NM_052961, NM_052978, NM_054021, NM_054108, NM_058165, NM_078485, NM_080552, NM_080617, NM_080671, NM_080742, NM_130773, NM_130900, NM_133180, NM_133266, NM_133338, NM_133339, NM_133340, NM_133341, NM_133342, NM_133343, NM_133344, NM_133489, NM_133493, NM_133642, NM_138290, NM_138718, NM_138996, NM_139204, NM_139316, NM_144497, NM_144725, NM_145725, NM_145726, NM_152562, NM_152854, NM_153819, NM_170696, NM_170697, NM_170775, NM_171827, NM_172337, NM_173479, NM_174869, NM_175052, NM_175611, NM_175709, NM_175768, NM_176095, NM_176096, NM_177555, NM_177556, NM_177557, NM_177558, NM_177959, NM_178154, NM_178155, NM_178156, NM_178157, NM_181457, NM_181458, NM_181459, NM_181460, NM_181461, NM_181466, NM_181467, NM_181468, NM_181469, NM_181505, NM_181657, NM_181689, NM_182609, NM_198265, NM_198287, NM_198407, NM_198570, NM_198849, NM_199051, NM_199076, NM_199077, NM_199231, NM_199234, NM_199425, NM_199426, NM_199427, NM_199427, NM_201647, NM_201999, NM_206827, NM_206866, NM_206961, NM_207121, NM_213622, NP 536846, and NR 002196.

18. The method of claim 9 , wherein the at least one marker DNA is selected from the group consisting of vimentin, BMP3, Septin 9, TFPI2, 2 regions of LRAT, and EYA4 DNAs.

19. A method of making an assay for characterizing DNA amplified from a methylated marker DNA in the presence of DNA amplified from the unmethylated marker DNA that exhibits background methylation, the method comprising:

a) isolating marker DNA from a first group of samples in which the marker DNA is unmethylated DNA exhibiting background methylation and from a second group of samples in which the marker DNA is methylated;

b) treating the isolated marker DNA under conditions wherein unmethylated cytosine (C) bases are converted to uracil (U) bases to produce treated isolated marker DNA;

c) processing the treated isolated marker DNA using digital sequencing and/or digital polymerase chain reaction (PCR) to detect unconverted C bases in three or more CpG loci in each of at least 1000 individual copies of the treated isolated marker DNA from each of the first and second groups of samples;

d) for each of the three or more CpG loci, determining a ratio between a mean unconverted C in that CpG locus in the first group of samples to a mean unconverted C in the corresponding CpG locus in the second group of samples;

e) selecting a subset of at least three CpG loci for which the percentage of individual copies of the treated isolated marker DNA from the first group of samples that have unconverted C in all of the at least three CpG loci in the subset is less than the percentage of individual copies of the marker DNA from the second group of samples that have unconverted C in all of the at least three CpG loci in the subset; and

f) creating a detection assay that detects the presence or absence of unconverted C in all of the at least three CpG loci in the subset of CpG loci in a strand of treated marker DNA, wherein the absence of unconverted C in any one of the CpG loci in the subset of CpG loci in a strand of the marker DNA produces an assay result classifying that strand of the marker DNA as not methylated,

wherein creating the detection assay comprises synthesizing at least one oligonucleotide complementary to a segment of nucleic acid comprising the subset of CpG loci.