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

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 highlight its potential for clinical applications, including 5mC biomarker detection and early disease diagnosis. Competing Interest Statement C.H. is a scientific founder, a member of the scientific advisory board and equity holder of Aferna Bio, AllyRNA, and Ellis Bio, a scientific cofounder and equity holder of Accent Therapeutics, and a member of the scientific advisory board of Rona Therapeutics and Element Biosciences. Q.D. holds equity in Ellis Bio Inc. R.L. serves as scientific advisor to Ellis Bio Inc. The remaining authors declare no competing interests. Footnotes In this revised version, we have refined the manuscript title to maintain scientific clarity while avoiding reference to specific commercial brands. We also made minor editorial adjustments throughout the text to improve readability and consistency. No changes were made to the data, results, or main conclusions of the study. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE294197