Systematic errors in enzymatic conversion limit cell-free DNA methylation specificity

Abstract Enzymatic methylation sequencing (EM-seq) converts unmethylated cytosines to uracils while preserving DNA integrity, making it attractive for liquid biopsies. Here we report a reproducible fragment-level over-conversion error in EM-seq, which is not observed in bisulfite-based conversion or in Oxford Nanopore sequencing. While bisulfite and nanopore errors occur at sporadic CpGs, EM-seq generates molecules that appear fully unmethylated, introducing a false-positive background signal that severely limits deconvolution specificity in cfDNA analysis.