Epigenetic analysis of cell-free DNA by fragmentomic profiling

Proc Natl Acad Sci U S A. 2022 Nov;119(44):e2209852119. doi: 10.1073/pnas.2209852119. Epub 2022 Oct 26.


Cell-free DNA (cfDNA) fragmentation patterns contain important molecular information linked to tissues of origin. We explored the possibility of using fragmentation patterns to predict cytosine-phosphate-guanine (CpG) methylation of cfDNA, obviating the use of bisulfite treatment and associated risks of DNA degradation. This study investigated the cfDNA cleavage profile surrounding a CpG (i.e., within an 11-nucleotide [nt] window) to analyze cfDNA methylation. The cfDNA cleavage proportion across positions within the window appeared nonrandom and exhibited correlation with methylation status. The mean cleavage proportion was ∼twofold higher at the cytosine of methylated CpGs than unmethylated ones in healthy controls. In contrast, the mean cleavage proportion rapidly decreased at the 1-nt position immediately preceding methylated CpGs. Such differential cleavages resulted in a characteristic change in relative presentations of CGN and NCG motifs at 5' ends, where N represented any nucleotide. CGN/NCG motif ratios were correlated with methylation levels at tissue-specific methylated CpGs (e.g., placenta or liver) (Pearson's absolute r > 0.86). cfDNA cleavage profiles were thus informative for cfDNA methylation and tissue-of-origin analyses. Using CG-containing end motifs, we achieved an area under a receiver operating characteristic curve (AUC) of 0.98 in differentiating patients with and without hepatocellular carcinoma and enhanced the positive predictive value of nasopharyngeal carcinoma screening (from 19.6 to 26.8%). Furthermore, we elucidated the feasibility of using cfDNA cleavage patterns to deduce CpG methylation at single CpG resolution using a deep learning algorithm and achieved an AUC of 0.93. FRAGmentomics-based Methylation Analysis (FRAGMA) presents many possibilities for noninvasive prenatal, cancer, and organ transplantation assessment.

Keywords: cancer detection; epigenetics; fragmentomics; liquid biopsy; noninvasive prenatal testing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biomarkers, Tumor / genetics
  • Cell-Free Nucleic Acids* / genetics
  • Cytosine
  • DNA / genetics
  • DNA Methylation
  • Epigenesis, Genetic
  • Female
  • Guanine
  • Humans
  • Liver Neoplasms* / genetics
  • Nucleotides
  • Phosphates
  • Pregnancy


  • Cell-Free Nucleic Acids
  • Biomarkers, Tumor
  • DNA
  • Cytosine
  • Guanine
  • Nucleotides
  • Phosphates