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

Loyfer, N.; Magenheim, J.; Darwish, A.; Isaac, S.; Ganbat, J.; Babikir, H.; Jhutty, A.; Wan, J.; Bayes-Genis, A.; Revuelta-Lopez, E.; Eden, A.; Solanki, R.; Dor, Y.; Kaplan, T.

doi:10.64898/2026.03.24.713040

bioRxiv · 10.64898/2026.03.24.713040

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

Loyfer, N.·Magenheim, J.·Darwish, A.·Isaac, S.·Ganbat, J.·Babikir, H.·Jhutty, A.·Wan, J.·Bayes-Genis, A.·Revuelta-Lopez, E.·Eden, A.·Solanki, R.·Dor, Y.·Kaplan, T.

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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.

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Loyfer, N., Magenheim, J., Darwish, A., Isaac, S., Ganbat, J., Babikir, H., Jhutty, A., Wan, J., Bayes-Genis, A., Revuelta-Lopez, E., Eden, A., Solanki, R., Dor, Y., Kaplan, T.. 2026-03-26. Systematic errors in enzymatic conversion limit cell-free DNA methylation specificity. https://doi.org/10.64898/2026.03.24.713040

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