# RECAP-seq: restriction enzyme-based CpG-methylated fragment amplification for early cancer detection

## TL;DR

RECAP-seq is a new method that detects early cancer by focusing on DNA methylation patterns in cell-free DNA, achieving high accuracy even at low cancer DNA levels.

## Contribution

RECAP-seq introduces a novel approach to enrich hypermethylated DNA fragments using restriction enzymes, enabling sensitive early cancer detection.

## Key findings

- RECAP-seq successfully detected cancer-specific signals at as low as 0.001% tumor DNA fraction.
- The method identified 7,091 hypermethylated markers, including ALX4, which correlates with colorectal cancer progression.
- Clinical validation showed 78.7% sensitivity and 95% specificity with an AUC of 0.932 in colorectal cancer detection.

## Abstract

Aberrant DNA methylation drives cancer development, yet current screening methods require substantial resources for targeted enrichment across multiple CpG-rich regions. Early cancer detection in cell-free DNA (cfDNA) presents additional challenges due to low circulating tumor DNA fractions (0.01–10%) that dilute cancer-specific signals. To address these limitations, we developed Restriction Enzyme-based CpG-methylated fragment AmPlification sequencing (RECAP-seq) to selectively enrich hypermethylated fragments from existing Enzymatic Methyl-seq (EM-seq) libraries. RECAP-seq combines EM-seq library preparation with BstUI restriction enzyme digestion to target CGCG motifs, achieving preferential enrichment of CpG islands. With spike-in experiments using cell line mixtures, RECAP-seq successfully distinguished samples as low as 0.001%. The method identified 7,091 hypermethylated markers, including ALX4 which showed progressive increases with colorectal cancer stage. Clinical validation using cfDNA from 35 healthy donors and 47 colorectal cancer patients demonstrated robust detection with an area under the curve (AUC) of 0.932, achieving 78.7% sensitivity at 95% specificity.

The online version contains supplementary material available at 10.1038/s41598-025-24708-y.