TISSUE-SPECIFIC METHYLATION MARKER
Provided herein are compositions comprising tissue-specific markers for identifying a tissue of origin of a cell-free nucleic acid, e.g., a cell-free DNA molecule. Also provided herein are methods, compositions, and systems for identifying a tissue of origin of a cell-free nucleic acid by determining an absolute amount of cell-free nucleic acids comprising the tissue-specific marker. Also provided herein are methods, compositions, and systems for detecting a cancer in a tissue of an organism by analyzing tissue-specific markers.
1 . A method of measuring cytosine methylation at one or more differentiated methylated regions (DMRs), the method comprising:
(a) obtaining cell-free DNA molecules from a first biological sample of a subject; and
(b) performing an assay on the cell-free DNA molecules to measure, for each of the one or more DMRs, an amount of the cell-free DNA molecules comprising a first methylation status of a target sequence in the DMR;
wherein (i) the one or more DMRs comprise a target sequence of one or both of Sestrin 3 (SESN3) and protein tyrosine kinase 2 beta (PTK2B); and (ii) the target sequence of each of the one or more DMRs comprises one or more CpG sites.
2 .- 4 . (canceled)
5 . The method of claim 1 , wherein the assay comprises hybridizing the cell-free DNA molecules comprising the target sequence to probes.
6 .- 10 . (canceled)
11 . The method of claim 1 , wherein the assay comprises amplifying the cell-free DNA molecules using one or more pairs of primers.
12 .- 15 . (canceled)
16 . The method of claim 1 , wherein the assay comprises bisulfite conversion of unmethylated cytosine residues in the cell-free DNA molecules to uracil.
17 . The method of claim 1 , wherein the assay comprises performing methylation-aware sequencing of cell-free DNA molecules from the first biological sample.
18 . The method of claim 1 , wherein the target sequence comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 CpG methylation sites.
19 . (canceled)
20 . The method of claim 1 , wherein the first methylation status comprises methylation density for individual sites within the target sequence, a distribution of methylated/unmethylated sites over a contiguous region within the target sequence, or a pattern or level of methylation for each individual methylation site within the target sequence.
21 . The method of claim 1 , wherein the target sequence comprises a higher methylation density in a first tissue of the subject as compared to a second tissue of the subject.
22 . (canceled)
23 . The method of claim 21 , wherein the target sequence comprises a methylation density in the first tissue that is more than 50%.
24 .- 30 . (canceled)
31 . The method of claim 1 , wherein the target sequence comprises a polynucleotide sequence of PTK2B having at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 1.
32 . The method of claim 1 , wherein the assay comprises (i) amplification using a primer comprising SEQ ID NO: 2, a primer comprising SEQ ID NO: 3, or both, or (ii) use of a detectably-labeled probe comprising SEQ ID NO: 4 for detection of the target sequence.
33 . The method of claim 1 , wherein the assay comprises (i) amplification using a primer comprising SEQ ID NO: 5, a primer comprising SEQ ID NO: 6, or both, or (ii) use of a detectably-labeled probe comprising SEQ ID NO: 7 for detection of the target sequence.
34 . The method of claim 1 , wherein the target sequence comprises a polynucleotide sequence of SESN3 having at least 60% identity to SEQ ID NO: 8.
35 . The method of claim 1 , wherein the assay comprises (i) amplification using a primer comprising SEQ ID NO: 9, a primer comprising SEQ ID NO: 10, or both, or (ii) use of a detectably-labeled probe comprising SEQ ID NO: 11 for detection of the target sequence.
36 . The method of claim 1 , wherein the assay comprises (i) amplification using a primer comprising SEQ ID NO: 12, a primer comprising SEQ ID NO: 13, or both, or (ii) use of a detectably-labeled probe comprising SEQ ID NO: 14 for detection of the target sequence.
37 .- 56 . (canceled)
57 . The method of claim 1 , wherein the target sequence of each of the one or more DMRs comprises an exon sequence.
58 . The method of claim 1 , wherein the one or more DMRs comprise a target sequence comprising one or more CpG sites of SEQ ID NO: 1.
59 . The method of claim 1 , wherein the one or more DMRs comprise a target sequence comprising one or more CpG sites of SEQ ID NO: 8.
60 . The method of claim 1 , wherein the measured amount of the cell-free DNA molecules comprising a first methylation status is an absolute amount.
61 . The method of claim 1 , wherein the cell-free DNA molecules comprising the first methylation status of the target sequence in the DMR comprise DNA molecules released from a site of cancer metastasis.