Single-cell DNA methylation profiling currently suffers from excessive noise and/or limited cellular throughput. We developed scTAM-seq, a targeted bisulfite-free method for profiling up to 650 CpGs in up to 10,000 cells per experiment, with a dropout rate as low as 7%. We demonstrate that scTAM-seq can resolve DNA methylation dynamics across B-cell differentiation in blood and bone marrow, identifying intermediate differentiation states that were previously masked. scTAM-seq additionally queries surface-protein expression, thus enabling integration of single-cell DNA methylation information with cell atlas data. In summary, scTAM-seq is a high-throughput, high-confidence method for analyzing DNA methylation at single-CpG resolution across thousands of single cells.

Single-cell DNA methylation profiling currently suffers from excessive noise and/or limited cellular throughput. We developed scTAM-seq, a targeted bisulfite-free method for profiling up to 650 CpGs in up to 10,000 cells per experiment, with a dropout rate of less than seven percent. scTAM-seq focuses sequencing coverage on informative, variably methylated CpGs that in many tissues and cell types make up a minor fraction of all CpGs. By applying scTAM-seq to B cells from blood and bone marrow, we demonstrate that it can resolve DNA methylation dynamics across B-cell differentiation at unprecedented resolution, identifying intermediate differentiation states that were previously masked. scTAM-seq additionally queries surface protein expression and somatic mutations, thus enabling integration of single-cell DNA methylation information with cell atlas data, and opening applications in tumor profiling. In summary, scTAM- seq is the first high-throughput, high-confidence method for analyzing DNA methylation at single-CpG resolution across thousands of single cells.