# Naturally-occurring DNA fragment termini correlate with methylation at CpG sites in hair and blood plasma cell-free DNA

## TL;DR

DNA fragmentation patterns in hair and blood plasma correlate with methylation at CpG sites, enabling methylation estimation from fragment termini.

## Contribution

A novel model for estimating CpG methylation using DNA fragment termini in hair and plasma cell-free DNA is developed.

## Key findings

- Methylation-dependent fragmentation occurs in hair and urine cell-free DNA.
- Using 5’ and 3’ termini improves CpG methylation modeling from sequence data.
- The model allows genotype and epigenetic inference from the same input data.

## Abstract

CpG methylation is an important epigenetic regulator in growth, development, and disease and a biomarker of age. It was recently shown that CpG methylation influences nuclease activity in the natural process that generates blood plasma cell-free DNA fragments. This results in a correlation between fragmentation sites and methylation that can be used to estimate methylation levels at CpG sites.

We find methylation-dependent fragmentation is also present in the process that generates the cell-free DNA fragments found in hair shafts and urine. Analysis of DNA fragments that includes accurate 5’ and 3’ termini and local sequence context improves modelling of CpG methylation from sequence data.

We demonstrate the existence of methylation-sensitive DNA fragmentation in rootless hair. We develop a model for estimating CpG methylation using both 5-prime and 3-prime native termini of these naturally occurring DNA fragments. This approach enables genotype and epigenetic inference from that same input data. It is applicable for samples that are prevalent in the field of liquid biopsy, forensics and ancient DNA and likely in other keratinized tissues where nuclease digestion of DNA occurs as part of regular development.

The online version contains supplementary material available at 10.1186/s12864-025-12459-z.