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Ultra-mild bisulfite outperforms existing methods for 5-methylcytosine detection with low input DNA

Qing Dai,Tanner Baldwin,Ruitu Lyu,B. Daniels,Chang Ye,Chen Cao,Chenyou Zhu,Diwen Fan,Liane Lin,Yushuai Liu,Yiding Wang,Chuan He

Published 2025 in Nature Communications

ABSTRACT

We present Ultra-Mild Bisulfite Sequencing (UMBS-seq), a method for 5-methylcytosine (5mC) detection that minimizes DNA degradation and background noise. UMBS-seq outperforms conventional bisulfite and enzymatic methyl-sequencing (EM-seq) methods in library yield, complexity, and conversion efficiency when applied to low-input DNA samples. In particular, its effectiveness with low-input cell-free DNA (cfDNA) and hybridization-based target capture highlights its potential for clinical applications, including 5mC biomarker detection and early disease diagnosis. DNA methylation is a critical biomarker for disease detection, yet current methods often compromise DNA integrity or lack sufficient accuracy. Here, the authors introduce Ultra-Mild Bisulfite Sequencing, a technique that enables highly sensitive, robust, and accurate detection of 5mC.

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