Immobilized solid-phase DNA deamination for epigenetic sequencing of sparse DNA samples such as cell-free DNA.

 Epigenetic modifications have been implicated in cancer and other indications, leading to the evolution of epigenetic sequencing methodologies to detect and analyze such modifications.  Current epigenetic sequencing methods frequently employ DNA deaminases or other DNA modifying enzymes; however, such methods are limited by multiple purification steps which lead to loss of input DNA and the need for single-stranded DNA, a difficult to generate substrate.

 The inventors developed a method to perform enzymatic deamination-based epigenetic sequencing on a solid phase to sequence rare, low count, DNA samples. The conjugation of DNA to solid-phase allows for greater DNA sequencing yields and analysis of rarer DNA samples such as cell-free DNA.

 Target DNA to be sequenced is ligated to adaptors which are resistant to enzymatic deamination due to the inclusion of a modified cytosine base.  Target DNA is also tagged with a 3’-biotin to immobilize on a solid phase containing streptavidin to minimize DNA loss due to processing and allow for denaturation of DNA on resin. In some applications, on resin deamination converts cytosine, but not 5-methylcytosine (5mC), into uracil, which allows for the distinction of methylated and unmethylated DNA. The process can be similarly adapted to detect 5-hydroxymethylcytosine (5hmC) or both 5mC and 5hmC.

  -  Immobilization minimizes DNA loss during processing and purification steps, allowing for sequencing of sparse samples
  -  DNA can be denatured on resin, reducing the need for single-strand DNA preparation.
  -  The same DNA strands can potentially be processed by more than one sequencing pipeline to detect different modifications on the same DNA molecule.
  -  Applicable to wide variety of deaminase-based DNA-sequencing pipelines.
  -  Streamlined sequencing library generation.