Methods for the epigenetic analysis of DNA, particularly cell-free DNA
Inventors
Arensdorf, Patrick A. • Spacek, Damek
Assignees
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Publication Number
US-11634748-B2
Publication Date
2023-04-25
Expiration Date
Abstract
Methods are provided for the epigenetic analysis of cell-free DNA using organic boranes to convert oxidized 5-methylcytosine residues in the cell-free DNA to dihydrouracil (DHU) residues. Cell-free DNA is contacted with an organic borane selected to successively bring about reduction, deamination, and decarboxylation of oxidized 5-methylcytosine residues such as 5-carboxymethylcytosine and 5-formylcytosine, resulting in DHU residues in place thereof. Following amplification, the treated cell-free DNA is sequenced, with the DHU residues read as thymine residues. Reaction mixtures, kits and additional methods are also provided, as are related methods for the epigenetic analysis of DNA, including cell-free DNA.
Core Innovation
The invention provides an epigenetic analysis of cell-free DNA in which 5hmC locations are identified in adapter-ligated target DNA. The approach oxidizes 5hmC in the target DNA with an oxidizing reagent to yield DNA containing oxidized 5hmC without affecting 5mC, wherein the oxidized 5hmC is selected from 5caC, 5fC, and combinations thereof.
The oxidized 5hmC is then reacted with pyridine borane to reduce, deaminate, and either decarboxylate or deformylate the oxidized 5hmC, thereby providing modified DNA containing DHU in place of the oxidized 5hmC. DHU is read as thymine during sequencing, enabling 5hmC-indicative sequence reads when amplified and sequenced.
The method further includes comparing the 5hmC-indicative sequence reads to standard sequence reads obtained for the target molecule, where a change from C in the standard sequence reads to T in the 5hmC-indicative sequence reads indicates a 5hmC location. The described workflow is bisulfite-free and without intermediate isolation, and is compatible with TET-assisted oxidation.
Claims Coverage
1 independent claim defines a bisulfite-free method for identifying 5hmC locations in adapter-ligated target DNA from a cell-free DNA sample using an oxidizing reagent plus pyridine borane conversion to DHU, followed by amplification/sequencing and C-to-T calling. Dependent claims add refinements concerning the absence of bisulfite, performing steps without isolating intermediates, specifying a particular oxidizing reagent type, constraining input to double-stranded DNA, and adding adapter-based sample and molecular barcode components.
Oxidation of 5hmC to oxidized forms without affecting 5mC
Oxidizing 5hmC in the target DNA with an oxidizing reagent to yield DNA containing oxidized 5hmC without affecting 5mC, wherein the oxidized 5hmC is selected from 5caC, 5fC, and combinations thereof.
Pyridine borane conversion of oxidized 5hmC to DHU-containing modified DNA
Reacting the DNA containing oxidized 5hmC with pyridine borane to reduce, deaminate, and either decarboxylate or deformylate the oxidized 5hmC, thereby providing modified DNA containing DHU in place of the oxidized 5hmC.
Sequencing and 5hmC-indicative read generation by DHU-to-thymine signal
Amplifying and sequencing the modified DNA to provide 5hmC-indicative sequence reads.
C-to-T comparison for identifying 5hmC locations
Comparing the 5hmC-indicative sequence reads to standard sequence reads obtained for the target molecule, wherein a change from C in the standard sequence reads to a T in the 5hmC-indicative sequence reads indicates a 5hmC location.
Absence of bisulfite during conversion steps (a) through (c)
Performing steps (a) through (c) in the absence of bisulfite.
No intermediate isolation across steps (a) through (c)
Performing steps (a) through (c) without isolating any intermediates.
Perruthenate-salt oxidizing reagent
Using a chemical oxidizing reagent that is a perruthenate salt.
Adapter ligation with sample identifier and molecular barcodes
Including adapter-ligated DNA that has adapters carrying a sample identifier sequence plus at least one additional molecular barcode selected from a fragment identifier sequence and a strand identifier sequence.
Cell-free DNA includes double-stranded DNA
The cell-free DNA includes double-stranded DNA.
Across the independent claim and its dependents, the core inventive concept is the oxidation of 5hmC to selected oxidized forms followed by pyridine borane conversion to DHU-containing modified DNA, then amplification/sequencing and identifying 5hmC by comparing standard reads to 5hmC-indicative reads based on a C-to-T change. Dependent claims further narrow the workflow by requiring absence of bisulfite, avoiding intermediate isolation, optionally specifying a perruthenate-salt oxidizing reagent, constraining input to double-stranded cell-free DNA, and adding adapter-based sample identifiers and fragment/strand molecular barcodes.
Stated Advantages
Bisulfite-free analysis using pyridine borane conversion and sequencing.
One-pot/one-tube conversion without intermediate isolation, as stated in the description chunk.
Compatibility with TET-assisted oxidation, as stated in the description chunk.
Selectivity demonstrated by mass/NMR examples for conversion of oxidized 5caC/5fC.
Documented Applications
Identifying 5hmC locations in adapter-ligated target DNA derived from a cell-free DNA sample, using amplification and sequencing read comparison.
Detecting and locating 5mC and 5hmC in the broader described workflow, including embodiments with enrichment/affinity tagging and co-occurrence on single strands.
Using molecular/process barcoding in conjunction with adapter-ligated DNA, including sample identifier sequences plus molecular barcodes (fragment and/or strand identifiers), as described in the chunk.
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