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

Area of Science:

• Molecular Biology
• Genomics
• Cancer Research

Background:

• Aberrant DNA methylation is a key driver of cancer development.
• Current screening methods for cancer DNA are resource-intensive and struggle with low circulating tumor DNA fractions.
• Detecting early cancer signals in cell-free DNA (cfDNA) is challenging due to signal dilution.

Purpose of the Study:

• To develop a novel method for selective enrichment of hypermethylated DNA fragments from cfDNA.
• To improve the sensitivity and efficiency of early cancer detection using DNA methylation markers.
• To address the limitations of existing enrichment strategies for cancer screening.

Main Methods:

• Developed Restriction Enzyme-based CpG-methylated fragment Amplification sequencing (RECAP-seq).
• Integrated RECAP-seq with Enzymatic Methyl-seq (EM-seq) library preparation and BstUI restriction enzyme digestion.
• Utilized spike-in experiments with cell line mixtures to assess sensitivity, down to 0.001% detection limits.

Main Results:

• RECAP-seq successfully enriched hypermethylated fragments, targeting CpG islands via CGCG motifs.
• Identified 7,091 hypermethylated markers, including ALX4, with stage-dependent increases in colorectal cancer.
• Clinical validation in cfDNA demonstrated robust colorectal cancer detection (AUC 0.932), achieving 78.7% sensitivity at 95% specificity.

Conclusions:

• RECAP-seq offers a sensitive and specific method for early cancer detection using cfDNA methylation.
• The technique overcomes challenges associated with low tumor DNA fractions and resource-intensive enrichment.
• RECAP-seq holds promise for advancing non-invasive cancer screening and diagnosis.