METHODS FOR DISEASE DETECTION
The present disclosure provides, among other things, methods for cancer detection (e.g., screening) and compositions related thereto. In various embodiments, the present disclosure provides methods for colorectal and/or advanced adenoma detection (e.g., screening) and compositions related thereto. In various embodiments, the present disclosure provides methods for screening that include analysis of methylation status of one or more methylation biomarkers, and compositions related thereto. In various embodiments, the present disclosure provides methods for detection (e.g., screening) that include detecting (e.g., screening) methylation status of one or more methylation biomarkers in cfDNA, e.g., in ctDNA. In various embodiments, the present disclosure provides methods for screening that include detecting (e.g., screening) methylation status of one or more methylation biomarkers in cfDNA, e.g., in ctDNA, using next-generation sequencing techniques.
1 . A method of detecting advanced adenoma in a human subject, the method comprising:
determining a methylation status of each of two or more markers identified in DNA from a sample obtained from the subject, and
determining whether the subject has advanced adenoma based at least in part on the determined methylation status of each of the two or more markers,
wherein each of at least two of the two or more markers is a methylation locus comprising at least a portion of a differentially methylated region (DMR) selected from the DMRs of FIGS. 3 A- 3 G , each said portion comprising at least three (3) CpGs and each said methylation locus having a length equal to or less than 5000 bp.
2 . The method of claim 1 , wherein the method comprises determining a methylation status of at least a portion of each of the following DMRs of Table 15:
chr
start
end
SEQ ID NO.
7
100785927
100786167
SEQ ID NO.: 221
14
97412990
97413410
SEQ ID NO.: 374
wherein each said portion comprises at least three (3) CpGs.
3 . The method of claim 1 , wherein the method comprises determining a methylation status of at least a portion of each of the following DMRs of Table 16:
chr
start
end
SEQ ID NO.
7
100785927
100786167
SEQ ID NO.: 221
14
97412990
97413410
SEQ ID NO.: 374
20
3083167
3083587
SEQ ID NO.: 411
8
37797956
37798676
SEQ ID NO.: 329
wherein each said portion comprises at least three (3) CpGs.
4 . The method of claim 1 , wherein the method comprises determining a methylation status of at least a portion of each of the following DMRs of Table 17:
chr
start
end
SEQ ID NO.
7
100785927
100786167
SEQ ID NO.: 221
14
97412990
97413410
SEQ ID NO.: 374
20
3083167
3083587
SEQ ID NO.: 411
8
37797956
37798676
SEQ ID NO.: 329
16
57091834
57092014
SEQ ID NO.: 387
4
7940020
7940200
SEQ ID NO.: 287
19
40811045
40811585
SEQ ID NO.: 403
1
154567391
154567691
SEQ ID NO.: 246
14
105364294
105364612
SEQ ID NO.: 376
9
61862430
61863030
SEQ ID NO.: 338
wherein each said portion comprises at least three (3) CpGs.
5 . The method of claim 1 , wherein the sample is a tissue sample, a blood sample, a stool sample, or a blood product sample.
6 . The method of claim 1 , wherein the sample comprises DNA that is isolated from blood or plasma of the human subject.
7 . The method of claim 1 , wherein the DNA is cell-free DNA (cfDNA) of the human subject.
8 . The method of claim 1 , wherein the method comprises determining the methylation status of each of the one or more markers using next generation sequencing (NGS).
9 . The method of claim 1 , wherein the method comprises using one or more capture baits that enrich for a target region to capture one or more corresponding methylation locus/loci.
10 . The method of claim 1 , wherein each methylation locus is equal to or less than 3000 bp in length.
11 .- 12 . (canceled)
13 . A method of detecting colorectal cancer in a human subject, the method comprising:
determining a methylation status of each of two or more markers identified in DNA from a sample obtained from the subject, and
determining whether the subject has colorectal cancer based at least in part on the determined methylation status of each of the two or more markers,
wherein each of at least two of the two or more markers is a methylation locus comprising at least a portion of a differentially methylated region (DMR) selected from the DMRs of FIG. 2 , each said portion comprising at least three (3) CpGs and each said methylation locus having a length equal to or less than 5000 bp.
14 . The method of claim 13 , wherein the method comprises determining a methylation status of at least a portion of each of the following DMRs of Table 8:
chr
start
end
SEQ ID NO.
7
96997902
96999222
SEQ ID NO.: 92
8
96145538
96145718
SEQ ID NO.: 108
each said portion comprising at least three (3) CpGs.
15 . The method of claim 13 , wherein the method comprises determining a methylation status of at least a portion of each of the following DMRs of Table 9:
chr
start
end
SEQ ID NO.
7
96997902
96999222
SEQ ID NO.: 92
8
96145538
96145718
SEQ ID NO.: 108
2
100322218
100322818
SEQ ID NO.: 28
2
29115776
29116791
SEQ ID NO.: 17
each said portion comprising at least three (3) CpGs.
16 . The method of claim 13 , wherein the method comprises determining a methylation status of at least a portion of each of the following DMRs of Table 10:
chr
start
end
SEQ ID NO.
7
96997902
96999222
SEQ ID NO.: 92
8
96145538
96145718
SEQ ID NO.: 108
2
100322218
100322818
SEQ ID NO.: 28
2
29115776
29116791
SEQ ID NO.: 17
2
88765502
88766042
SEQ ID NO.: 25
4
153249541
153249721
SEQ ID NO.: 55
2
86790271
86790811
SEQ ID NO.: 24
2
176094518
176094878
SEQ ID NO.: 35
3
37453325
37453874
SEQ ID NO.: 41
each said portion comprising at least three (3) CpGs.
17 . The method of claim 13 , wherein the sample is a tissue sample, a blood sample, a stool sample, or a blood product sample.
18 . The method of claim 13 , wherein the sample comprises DNA that is isolated from blood or plasma of the human subject.
19 . The method of claim 13 , wherein the DNA is cell-free DNA (cfDNA) of the human subject.
20 . The method of claim 13 , wherein the method comprises determining the methylation status of each of the one or more markers using next generation sequencing (NGS).
21 . The method of claim 13 , wherein the method comprises using one or more capture baits that enrich for a target region to capture one or more corresponding methylation locus/loci.
22 . The method of claim 13 , wherein each methylation locus is equal to or less than 3000 bp in length.
23 . A method of detecting colorectal cancer in a human subject, the method comprising:
determining a methylation status for both of the following in deoxyribonucleic acid (DNA) from a sample of a human subject:
(i) a methylation locus within gene DLX6-AS1; and
(ii) a methylation locus within gene GDF6; and
diagnosing colorectal cancer in the human subject based on at least said determined methylation status.
24 . The method of claim 23 , comprising determining a methylation status for a methylation locus within gene DLX6-AS1, wherein the methylation locus within gene DLX6-AS1 comprises at least a portion of [chr7: 96997902-96999222] (SEQ ID NO.: 92), said portion comprising at least three (3) CpGs, wherein the methylation locus within gene DLX6-AS1 has a length equal to or less than 5000 bp.
25 . The method of claim 23 , comprising determining a methylation status for a methylation locus within gene GDF6, wherein the methylation locus within gene GDF6 comprises at least a portion of [chr8: 96145538-96145718](SEQ ID NO.: 108), said portion comprising at least three (3) CpGs, wherein the methylation locus within gene GDF6 has a length equal to or less than 5000 bp.
26 .- 205 . (canceled)
206 . The method of claim 1 , wherein the method comprises determining a methylation status of at least a portion of each of the following DMRs:
chr
start
end
SEQ ID NO.
14
97412990
97413410
SEQ ID NO.: 374
8
37797956
37798676
SEQ ID NO.: 329
16
57091834
57092014
SEQ ID NO.: 387
4
7940020
7940200
SEQ ID NO.: 287
1
154567391
154567691
SEQ ID NO.: 246
wherein each said portion comprises at least three (3) CpGs.
207 . The method of claim 13 , wherein the method comprises determining a methylation status of at least a portion of the DMR chr2:100322218-100322818 (SEQ ID NO.: 28), wherein said portion comprises at least three (3) CpGs.