ArticleClinical epigenetics2025
Comparison of enzymatic and bisulfite conversion of circulating cell-free tumor DNA for DNA methylation analyses.
Article in Clinical epigenetics, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 7 papers.
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Who cites it
7 citing papers in PubMed.
- Biological basis and clinical translation prospects of circulating cell-free DNA in precision management of breast cancer (Review).Oncology letters · 2026Review
- Circulating cell-free methylated DNA immunoprecipitation sequencing (cfMeDIP-seq) in oncology: from technological advances to clinical applications.NPJ precision oncology · 2026Review
- A New Complexity Layer: DNA Methylation and the Predictive Impact of Epigenetic Tests.International journal of molecular sciences · 2026Review
- Application of a modified MSRE-qPCR method for detecting circulating cell-free DNA methylation in cervical cancer.Frontiers in oncology · 2026Article
- Early detection of multiple cancers: the era of methylation-based liquid biopsy.Frontiers in oncology · 2026Review
- Review
- Comparison of enzymatic and bisulfite-based methods for sequencing-based cell-free DNA methylation profiling.Frontiers in epigenetics and epigenomics · 2025Article
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Authors and funding
6 authors.
Funding
Abstract
backgroundDetection of DNA methylation biomarkers in circulating cell-free DNA (cfDNA) has great clinical potential for cancer management, but there is a high need for method optimization and standardization. Bisulfite conversion of DNA is the gold-standard pre-treatment method for DNA methylation analyses, but causes also DNA fragmentation and loss. Enzymatic conversion of DNA represents a promising alternative due to the more gentle treatment minimizing damage to DNA. The aim of this study was to evaluate and compare enzymatic and bisulfite conversion to identify the best pre-treatment method for detecting DNA methylation biomarkers in cfDNA from plasma using droplet digital PCR (ddPCR).
resultsThe performance of the NEBNext Enzymatic Methyl-seq Kit (both the full kit intended for sequencing and the sub-component NEBNext Enzymatic Methyl-seq Conversion Module) and the EpiTect Plus DNA Bisulfite Kit was evaluated and compared using normal cfDNA and tumor cfDNA samples from colorectal cancer patients. The cytosine conversion efficiency was 99-100% for both enzymatic and bisulfite conversion. Enzymatic conversion resulted in longer DNA fragments with higher peak fragment sizes compared to bisulfite conversion, but the DNA recovery was considerably lower after enzymatic conversion (34-47%) compared to bisulfite conversion (61-81%). For enzymatic conversion, the full kit gave slightly better DNA recovery than the conversion module. A comparison of five magnetic bead brands, as well as several different magnetic bead-to-sample ratios revealed no major improvements in DNA recovery for the enzymatic conversion. DNA methylation of the biomarker BCAT1 was detected at similar rates in parallel tumor cfDNA samples pre-treated with either enzymatic or bisulfite conversion. However, enzymatic conversion resulted in lower number of positive droplets for both target and control ddPCR assays, in line with the lower DNA recovery after conversion.
conclusionsBased on a thorough evaluation of enzymatic and bisulfite conversion of cfDNA using ddPCR, bisulfite conversion emerges as the best pre-treatment method due to higher DNA recovery after conversion and higher number of positive droplets in the ddPCR reactions.
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