Methods of capturing cell-free methylated DNA and uses of same
There is described herein, a method of capturing cell-free methylated DNA from a sample having less than 100 mg of cell-free DNA, comprising the steps of: subjecting the sample to library preparation to permit subsequent sequencing of the cell-free methylated DNA; adding a first amount of filler DNA to the sample, wherein at least a portion of the filler DNA is methylated; denaturing the sample; and capturing cell-free methylated DNA using a binder selective for methylated polynucleotides.
1. A method of capturing cell-free methylated DNA from a sample having less than 100 ng of cell-free DNA, comprising the steps of:
a. subjecting the sample to library preparation to permit subsequent sequencing of the cell-free methylated DNA;
b. adding a first amount of filler DNA to the sample, wherein at least a portion of the filler DNA is methylated;
c. denaturing the sample; and
d. capturing cell-free methylated DNA using a binder selective for methylated polynucleotides;
wherein the cell-free DNA from the sample and the first amount of filler DNA together comprises at least 50 ng of total DNA.
2. The method of claim 1 further comprising the step of amplifying and subsequently sequencing the captured cell-free methylated DNA.
3. The method of claim 1 , wherein the sample has less than 50 ng of cell-free DNA.
4. The method of claim 1 , wherein the first amount of filler DNA comprises about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% methylated filler DNA with any remainder being unmethylated filler DNA.
5. The method of claim 1 , wherein the first amount of filler DNA is from 20 ng to 100 ng.
6. The method of claim 1 , wherein the cell-free DNA from the sample and the first amount of filler DNA together comprises at least 100 ng of total DNA.
7. The method of claim 1 , wherein the filler DNA is 50 bp to 800 bp long.
8. The method of claim 1 , wherein the filler DNA is double stranded.
9. The method of claim 1 , wherein the filler DNA is junk DNA.
10. The method of claim 1 , wherein the filler DNA is endogenous or exogenous DNA.
11. The method of claim 10 , wherein the filler DNA is non-human DNA.
12. The method of claim 1 , wherein the filler DNA is λ, DNA.
13. The method of claim 1 , wherein the binder is a protein comprising a Methyl-CpG-binding domain.
14. The method of claim 13 , wherein the protein is a MBD2 protein.
15. The method of claim 1 , wherein step (d) comprises immunoprecipitating the cell-free methylated DNA using an antibody.
16. The method of claim 15 , comprising adding at least 0.05 μg of the antibody to the sample for immunoprecipitation.
17. The method of claim 15 , wherein the antibody is 5-MeC antibody or 5-hydroxymethyl cytosine antibody.
18. The method of claim 15 , further comprising the step of adding a second amount of control DNA to the sample after step (b) for confirming the immunoprecipitation reaction.
19. The method of claim 1 , further comprising the step of adding a second amount of control DNA to the sample after step (b) for confirming the capture of cell-free methylated DNA.
20. A method of measuring a DNA methylation profile within the sample comprising the method of claim 1 .
21. A method of monitoring immune therapy or determining cell turnover comprising the method of claim 20 .
22. The method of claim 4 , wherein the first amount of filler DNA comprises between 5% and 50%, between 10% and 40%, or between 15% and 30% methylated filler DNA.
23. The method of claim 5 , wherein the first amount of filler DNA is from 30 ng to 100 ng, or from 50 ng to 100 ng.