Abstract: Disclosed herein include systems, machines, devices, and methods for single-pass methylation mapping. C-to-T converted sequence reads and G-to-A converted sequence reads generated from a sample subjected to a methylation assay can be mapped to a mapping reference sequence comprising a C-to-T converted reference sequence and a G-to-A converted reference sequence generated to a reference genome sequence. The counts of Cs and Ts of sequence reads mapped to each of one or more positions with Cs in the reference genome sequence can be used to determine whether the position is a methylated C or an unmethylated C in the sample.