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-11274335-B2
Publication Date
2022-03-15
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-carboxylcytosine 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 relates to epigenetic analysis of cell-free DNA at single-base resolution by detecting 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) residues through a non-bisulfite approach. Oxidized 5-methylcytosine derivatives, including 5-carboxylcytosine (5caC) and 5-formylcytosine (5fC), are converted into dihydrouracil (DHU), and after amplification and sequencing, DHU is read as thymine to enable base-resolution mapping of modified cytosine locations.
The disclosure frames bisulfite sequencing as problematic due to DNA degradation and incomplete conversion that can generate false positives. It therefore treats fractions of the same adapter-ligated, fragmented template DNA differently for selective handling of 5hmC versus 5mC, including blocking 5hmC in one fraction and enzymatically oxidizing 5mC to oxidized 5mC residues before organic borane chemistry converts those residues to DHU.
In a second fraction, 5hmC residues are oxidized to oxidized 5hmC residues selected from 5caC and 5fC without modifying 5mC residues, and those oxidized 5hmC residues are converted to DHU using pyridine borane. Sequencing both fractions and comparing sequence reads identifies which positions correspond to 5mC and which correspond to 5hmC, based on whether the modified residues read as cytosine or as thymine after DHU conversion.
Claims Coverage
The independent claim is clm-00001. It includes four inventive features covering a two-fraction method for selective blocking and oxidation, pyridine borane DHU conversion, paired sequencing, and comparative identification of 5mC and 5hmC locations.
Two-fraction detection for 5mC and 5hmC locations in adapter-ligated cfDNA
A method for detecting the locations of 5mC and 5hmC residues in a cell-free DNA sample comprising fragmented, adapter-ligated template DNA by processing a first fraction and a second fraction and comparing the resulting sequence reads to identify 5mC and 5hmC locations.
Blocking 5hmC in the first fraction and DHU conversion after 5mC oxidation
In the first fraction, blocking 5hmC residues with a blocking reagent to yield blocked 5hmC residues, enzymatically oxidizing 5mC residues to provide oxidized 5mC residues selected from 5caC, 5fC, and combinations thereof, converting the oxidized 5mC residues to DHU by treatment with pyridine borane, and amplifying and sequencing the first fraction DNA to provide first fraction sequence reads in which the blocked 5hmC residues read as C and DHU reads as T.
Selective oxidation of 5hmC in the second fraction while preserving 5mC and DHU conversion
In the second fraction, oxidizing 5hmC residues with an oxidizing reagent effective to convert 5hmC residues to oxidized 5hmC residues without modifying 5mC residues, wherein the oxidized 5hmC residues are selected from 5caC, 5fC, and combinations thereof, converting the oxidized 5hmC residues to DHU by treatment with pyridine borane, and amplifying and sequencing the second fraction DNA to provide second fraction sequence reads in which the unmodified 5mC residues read as C and DHU reads as T.
Comparing paired fraction reads to identify 5mC versus 5hmC locations
Comparing the first fraction sequence reads with the second fraction sequence reads to identify 5mC and 5hmC locations in the template DNA.
Independent claim clm-00001 centers on selectively treating two fractions of adapter-ligated cfDNA so that 5hmC and 5mC can be mapped by blocking 5hmC and oxidizing 5mC to DHU in the first fraction, selectively oxidizing 5hmC to DHU while preserving 5mC in the second fraction, and comparing the two sets of sequencing reads where blocked or unmodified residues read as C and DHU reads as T.
Stated Advantages
Enables discrimination of 5mC versus 5hmC while avoiding issues associated with bisulfite sequencing, which include DNA degradation and incomplete conversion that can lead to false positives.
Documented Applications
Epigenetic analysis of cell-free DNA at single-base resolution by detecting locations of 5mC and 5hmC residues in a fragmented, adapter-ligated sample.
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