The establishment and removal of DNA methylation are usually related to changes in gene expression during embryonic development and disease occurrence. With the advancement of NGS technology, whole-genome DNA methylation sequencing technology based on bisulfite conversion has become an important tool to demonstrate the genome-wide distribution of DNA methylation. Most of the emerging single-cell DNA methylation sequencing technologies are based on bisulfite reactions. However, bisulfite will cause fragmentation or even degradation of genomic DNA. Affected by this, single-cell DNA methylation sequencing based on bisulfite conversion has the disadvantage of low genome coverage. Due to bisulfite reaction cannot distinguish between 5mC and5hmC,it is difficult to accurately characterize DNA methylation status in 5hmC-enriched cell types such as pluripotent cell populations (early embryos, embryonic stem cells) and neuronal cells. Therefore, it is necessary to develop single-cell methylation and hydroxymethylation sequencing technologies with high genome coverage.

　　On November 27, 2023, PNAS published a research article entitled "Single-cell bisulfite-free 5mC and 5hmC sequencing with high sensitivity and scalability” from Prof. Xiaoliang Sunney Xie’s group, introducing a bisulfite-free single-cell 5mC and 5hmC sequencing technologynamed Cabernet (Carrier-Assisted Base-conversion by Enzymatic ReactioN with End-Tagging). This methodwas used to profile the changes of DNA methylation, hydroxymethylation and hemi-methylation,revealing the dynamics of active and passive demethylation during early mouse embryonic development.

　　Through the employment ofTn5 transposition and enzymatic conversion module from EM-seq (NEB), DNA from an individual cell could be processed C-to-T transition and specifically amplified with transposed adapters. By comparing with well-establishedtechnologies, the accuracy of Cabernet was verified, and it also showed advantages in terms of high mapping rate and high genome coverage. This study also introduced Cabernet-H technology, which enables single-cell DNA hydroxymethylation sequencing with high genome coverage. Meanwhile, the use of Tn5 transposition would result in different insertion sites on two alleles, which helps distinguish two alleles and measure hemi-methylation status (asymmetrical methylation status on the two DNA strands) in each allele.

　　The performance of Cabernet and Cabernet-H

　　This method was used to characterize the changes of methylation, hydroxymethylation and hemi-methylation during early mouse embryonic development, and revealthe dynamics of TET-mediated active demethylation activityand replication-dependent passive demethylation activity. In addition, this study also demonstrated the differences in the mechanisms involved in the regulation of parental gene expression by active and passive demethylation.

　　The dynamics of 5mC, 5hmC and hemi-methylation in the early embryos

　　By using combinatorial indexing, a high-throughput DNA methylation detection platform was developed. This method is cost-effective and can efficiently detect thousands of single-cell DNA methylation maps in two days.

　　High-throughput DNA methylation detection

　　Cao Yunlong (BIOPIC), Bai Yali (CPL), Yuan Tianjiao (BIOPIC)are the co-first authors of this paper. Prof. Xie Xiaoliang is the corresponding author. This study was supported by Changping Laboratory, Beijing Advanced Innovation Center for Genomics at Peking University, and the Beijing Municipal Commission of Science and Technology.

　　Article link:https://doi.org/10.1073/pnas.2310367120