Tools for Quantifying DNA Methylation and Heterogeneity in Cancer

Abstract

DNA methylation is a heritable epigenetic mark essential for a myriad of biological processes. Dysregulation of DNA methylation either by disruption of the methylation machinery, cellular environment, or replication rate contributes to diseases such as developmental disorders and cancer (Smith et al, 2013; Greenberg et al, 2019). Here we investigated methylation alterations in cellular models in the context of oncogenic mutations that are linked to hypermethylation in tumors. Our challenges in these efforts motivated technology development. We present ZEN-seq a novel method for targeted single-cell profiling of DNA methylation. Our design draws a balance between expanding coverage of regulatory elements and enriching for informative CpG dinucleotides. Barcoded DNA fragments are pooled prior to bisulfite conversion, allowing multiplex processing, improving consistency, and reducing cost and labor. Bisulfite-treated DNA is then amplified using primers with unique molecular identifiers, enabling library generation from highly fragmented and low input DNA samples. We further adapt the method for single-cell targeted methylation profiling, and demonstrate its applicability to evaluate methylation differences between cell types and cell-to-cell variability. Overall, our work provides a technology that can be applied to elucidating aberrant tumor DNA methylomes.