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End-repair causes methylation underestimation in cell-free DNA sequencing libraries

Groth, T. E.; Mishin, A. A.; Rao, V.; Tibet, R.; Troll, C. J.

2025-12-17 genomics
10.64898/2025.12.15.694439 bioRxiv
Show abstract

Cell-free DNA methylation sequencing provides insight into tissue of origin and chromatin structure. In some workflows, generating libraries includes end-repair. Using matched single-stranded and double-stranded libraries prepared from the same cfDNA extracts, we show that end-repair in double-stranded DNA libraries reduces globally inferred CpG methylation leading to decreased tissue of origin accuracy. Trimming read termini partially mitigates this bias but decreases coverage and removes fragmentomic information compared to single-stranded DNA libraries, which forego end-repair.

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