Granted Patent
B2
US 12,655,417 · App. 19/241,820 · Granted Jun 16, 2026
Methods of capturing cell-free methylated DNA and uses of same
Inventors:
Daniel Diniz De Carvalho (Toronto, CA); Shu Yi Shen (Markham, CA); Rajat Singhania (Toronto, CA)
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Abstract
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.
Claims (22)
1 . A method for processing a nucleic acid sample of a subject, comprising:
processing one or more nucleic acid molecules derived from said nucleic acid sample, wherein said nucleic acid sample has no more than 50 nanograms (ng) of nucleic acid molecules, wherein said one or more nucleic acid molecules comprise one or more cell-free nucleic acid molecules, and wherein said one or more cell-free nucleic acid molecules comprise one or more methylated cell-free nucleic acid molecules, under conditions sufficient to increase a fold enrichment ratio associated with said one or more methylated cell-free nucleic acid molecules.
2 . The method of claim 1 , wherein said nucleic acid sample is a cell-free deoxyribonucleic acid (cfDNA) sample.
3 . The method of claim 1 , wherein said one or more cell-free nucleic acid molecules comprise one or more unmethylated nucleic acid molecules.
4 . The method of claim 3 , wherein said processing comprises determining (1) a presence of one or more methylated regions of said one or more methylated cell-free nucleic acid molecules and (2) a presence of one or more unmethylated regions of said one or more unmethylated nucleic acid molecules.
5 . The method of claim 1 , wherein said fold enrichment ratio is a ratio as determined by a comparison of said one or more methylated cell-free nucleic acid molecules to one or more unmethylated cell-free nucleic acid molecules.
6 . The method of claim 1 , wherein said fold enrichment ratio is at least 25.
7 . The method of claim 1 , wherein said processing further comprises combining said one or more nucleic acid molecules derived from said nucleic acid sample with a plurality of filler nucleic acid molecules.
8 . The method of claim 1 , wherein said processing further comprises capturing said one or more methylated cell-free nucleic acid molecules using one or more binders.
9 . The method of claim 1 , further comprising sequencing said one or more methylated cell-free nucleic acid molecules or derivatives thereof, thereby generating a methylation profile of said subject.
10 . The method of claim 9 , further comprising processing one or more nucleic acid molecules derived from another nucleic acid sample from a healthy subject.
11 . The method of claim 10 , wherein said processing of said one or more nucleic acid molecules derived from said another nucleic acid sample further comprises enriching for one or more methylated nucleic acid molecules derived from said another nucleic acid sample.
12 . The method of claim 11 , further comprising sequencing said one or more methylated nucleic acid molecules derived from said another nucleic acid sample, thereby generating a methylation profile of said healthy subject.
13 . The method of claim 12 , further comprising comparing said methylation profile of said subject to said methylation profile of said healthy subject.
14 . A method for processing a nucleic acid sample of a subject, comprising:
processing one or more nucleic acid molecules derived from said nucleic acid sample to produce enriched nucleic acid molecules, wherein said one or more nucleic acid molecules comprise one or more cell-free nucleic acid molecules, under conditions sufficient to increase a CpG enrichment score as determined by dividing a CpG frequency of said enriched nucleic acid molecules by a CpG frequency of a human genome, wherein said CpG enrichment score is at least 2.
15 . The method of claim 14 , further comprising sequencing said enriched nucleic acid molecules or derivatives thereof, thereby generating a plurality of sequencing reads of said enriched nucleic acid molecules.
16 . The method of claim 15 , further comprising determining said CpG frequency of said enriched nucleic acid molecules based on said plurality of sequencing reads.
17 . The method of claim 14 , wherein one or more CpG regions of said enriched nucleic acid molecules comprise one or more methylated CpG regions.
18 . The method of claim 14 , wherein said CpG enrichment score is at least 3.
19 . The method of claim 1 , further comprising determining one or more characteristics of said one or more methylated cell-free nucleic acid molecules, thereby generating a methylation profile of said subject.
20 . The method of claim 14 , further comprising determining one or more characteristics of said enriched nucleic acid molecules, thereby generating a methylation profile of said subject.
Assignments (2)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Oct 7, 2025
From: DINIZ DE CARVALHO, DANIEL; SHEN, SHU YI; SINGHANIA, RAJAT
To: UNIVERSITY HEALTH NETWORK
Reel/Frame 072492/0832 →
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Oct 7, 2025
From: UNIVERSITY HEALTH NETWORK
To: UNIVERSITY HEALTH NETWORK; SINAI HEALTH SYSTEM
Reel/Frame 072493/0283 →
Continuity (6)
Continuation
16098620
Provisional Application
62331070
· May 3, 2016
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