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

• Published in: Clinical Epigenetics
• Main Authors: Barrett Nuttall, Daniel L. Karl, Kathleen Burke, Megan Callahan, Kerrin Mendler, Pablo Cingolani, Steven Criscione, Serhiy Naumenko, Elena Bibikova, Veerendra Munugalavadla, John C. Byrd, Richard R. Furman, Jennifer R. Brown, Andrew Mortlock, Brian A. Dougherty, J. Carl Barrett, Maurizio Scaltriti, James Hadfield
• Format: Article
• Language: English
• Published: BMC 2025-10-01
• Subjects: Methylation; Enzymatic methylation sequencing; Bisulfite sequencing; Targeted methylation sequencing; Chronic lymphocytic leukemia
• Online Access: https://doi.org/10.1186/s13148-025-01959-0

Abstract 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, we report a putative link of interleukin (IL)-15 methylation changes to acalabrutinib treatment response in a CLL clinical trial cohort (ACE-CL-001 trial, NCT02029443).