Selective labeling of 5-methylcytosine in circulating cell-free DNA
The present disclosure provides methods for selectively tagging 5-methylcytosine in a DNA sample and using this approach for genome-wide profiling of 5-methylcytosine in a low input DNA sample such as circulating cell-free DNA.
1. A method for creating a library of 5-methylcytosine (5mC) containing DNA from a low-input DNA sample comprising the steps in order of:
(a) obtaining a low-input DNA sample;
(b) adding a blocking group to the endogenous 5-hydroxymethylcytosine (5hmC) in the DNA sample;
(c) converting the 5mC in the DNA sample to 5hmC with Naeglerai gruberi TET1 (NgTET1);
(d) adding a linking group to the 5hmC from step (c);
(e) after step (d), ligating DNA adapters to the DNA sample;
(f) after step (e), adding an affinity tag to the linking group from step (d);
(g) enriching for the affinity tagged DNA from step (f) by affinity purification utilizing a support; and
(h) amplifying the enriched DNA from step (g), wherein amplifying the enriched DNA comprises PCR directly on the enriched DNA linked to the support.
2. The method of claim 1 , wherein the DNA sample comprises less than about 10 ng or less than about 5 ng of DNA.
3. The method of claim 1 , wherein the DNA sample comprises circulating cell-free DNA (cfDNA).
4. The method according to claim 1 , wherein the linking group is a modified glucose moiety and the step of adding a linking group to the 5hmC from step (c) comprises providing UDP linked to the modified glucose moiety in the presence of βGT.
5. The method of claim 4 , wherein the modified glucose moiety is a 6-azide-glucose moiety.
6. The method according to claim 1 , wherein the affinity tag comprises biotin and the affinity purification utilizes streptavidin linked to a support.
7. The method of claim 6 , wherein the step of adding the biotin affinity tag to the linking group comprises contacting the 5hmC modified with a linking group with biotin that comprises an azide-reactive group.
8. The method of claim 6 wherein the biotin comprises PEG-dibenzocyclooctyne (DBCO)-biotin.
9. The method according to claim 6 , wherein amplifying the enriched DNA comprises PCR directly on the enriched DNA linked to the support by the biotin-streptavidin.
10. The method of claim 1 , wherein the blocking group is glucose.
11. The method of claim 8 , wherein the glucose blocking group is added to the endogenous 5hmC by contacting the DNA sample with UDP-glucose in the presence of β-glycosyltransferase (βGT).
12. The method according to claim 1 , wherein steps (c) and (d) are performed as a one pot reaction.
13. A method for selectively sequencing 5mC containing DNA from a DNA sample comprising the steps in order of:
(a) adding a blocking group to the endogenous 5-hydroxymethylcytosine (5hmC) in the DNA sample;
(b) converting the 5mC in the DNA sample to 5hmC with Naegleria gruberi TET1 (NgTET1);
(c) adding a linking group to the 5hmC from step (b);
(d) after step (c), ligating adapter DNA to the DNA sample;
(e) after step (d), adding an affinity tag to the to the linking group from step (c);
(f) enriching for the affinity tagged DNA from step (e) by affinity purification utilizing a support;
(g) amplifying the enriched DNA from step (f), wherein amplifying the enriched DNA comprises PCR directly on the enriched DNA linked to the support; and
(h) sequencing the amplified DNA.
14. The method of claim 13 , wherein the DNA sample comprises less than about 10 ng or less than about 5 ng of DNA.
15. The method of claim 13 , wherein the DNA sample comprises circulating cell-free DNA (cfDNA).
16. The method according to claim 13 , wherein the linking group is a modified glucose moiety and the step of adding a linking group to the 5hmC from step (b) comprises providing UDP linked to the modified glucose in the presence of βGT.
17. The method of claim 16 , wherein the modified glucose moiety is a 6-azide-glucose moiety.
18. The method according to claim 13 , wherein the affinity tag comprises biotin and the affinity purification utilizes streptavidin linked to a support.
19. The method of claim 18 , wherein the step of adding the biotin tag to the linking group comprises contacting the 5hmC modified with a linking group with biotin that comprises an azide-reactive group.
20. The method of claim 18 wherein the biotin comprises PEG-dibenzocyclooctyne (DBCO)-biotin.
21. The method according to claim 18 , wherein amplifying the enriched DNA comprises PCR directly on the enriched DNA linked to the support by the biotin-streptavidin.
22. The method of claim 13 , wherein the blocking group is glucose.
23. The method of claim 22 , wherein the glucose blocking group is added to the endogenous 5hmC by contacting the DNA sample with UDP-glucose in the presence of β-glycosyltransferase (βGT).
24. The method according to claim 14 , wherein the concentration of the TET1 enzyme is less than 3 less than 2 or less than 1 μM.
25. The method according to claim 4 , wherein the concentration of βGT is less than about 2 μM, less than about 1 μM, less than 0.5 μM, less than about 0.3 μM, between about 0.1 μM to about 1 μM, between about 0.2 μM and about 0.5 μM, or about 0.3 μM.