Comprehensive comparison of enzymatic and bisulfite DNA methylation analysis in clinically relevant samples

作者：Barrett R. B. Nuttall, Daniel L. Karl, Kathleen A. Burke, Megan Callahan, Kerrin Mendler, Pablo Cingolani, Steven W. Criscione, Serhiy Naumenko, Елена Бибикова, Veerendra Munugalavadla, John C. Byrd, Richard R. Furman, Jennifer R. Brown, Andrew A. Mortlock, Brian Andrew Dougherty, J. Carl Barrett, Maurizio Scaltriti, James Hadfield · 发表于：Clinical Epigenetics · 年份：2025 · DOI：10.1186/s13148-025-01959-0 · 被引用次数：9 · 研究领域：Epigenetics and DNA Methylation、Acute Lymphoblastic Leukemia research、Acute Myeloid Leukemia Research

BACKGROUND: Bisulfite conversion is considered the gold standard for DNA methylation analysis, but it damages DNA and performs sub-optimally with clinical samples (e.g., formalin-fixed paraffin-embedded and circulating free plasma DNA (cfDNA)). Here we describe a comprehensive comparison of bisulfite and enzymatic methylation sequencing, using commercially available assays in clinically relevant patient samples and cell lines. We also report the first clinical enzymatic whole genome methylation sequencing (WGMS) in a cohort of patients with chronic lymphocytic leukemia (CLL). We report data from a multi-arm experiment comprising controlled reference material and clinically relevant samples to assess technical differences between enzymatic and chemical methylation conversion technologies. RESULTS: Enzymatic methylation sequencing was highly concordant to bisulfite data but outperformed bisulfite conversion in key sequencing metrics; the enzymatic method demonstrated significantly higher estimated counts of unique reads, reduced DNA fragmentation, and higher library yields than bisulfite conversion. Enzymatic conversion produced inferior methylation array data. Although bisulfite and enzymatic methods were highly concordant, the increased quality of multiple sequencing metrics seen in the enzymatic method enabled the development of robust clinical sample pipelines including targeted sequencing in cfDNA. CONCLUSIONS: Using the enzymatic methylation sequencing methods described, ...