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

Here we present Ultra-Mild Bisulfite Sequencing (UMBS-seq), which minimizes DNA damage and background noise while detecting 5-methylcytosine (5mC) in DNA. When applied to low-input lambda DNA (5 ng-10 pg), UMBS-seq achieved superior library yields, insert sizes, conversion efficiency, and CpG coverages compared to enzymatic alternatives. These results were recapitulated with low-input cell-free DNA (cfDNA) and in combination with targeted capture, highlighting the potential of UMBS-seq for future disease diagnosis. Overall design: Ultra-Mild Bisulfite Sequencing (UMBS-seq), EM-seq (NEB), and conventional bisulfite sequencing (Zymo Research) were performed using low-input lambda DNA and cell-free DNA ranging from 0.01 ng to 5 ng.