Complexity of WGBS Data Caused by Cellular Heterogeneity and Multiple Cytosine Modifications

DNA methylation is an important epigenetic modification that plays an important role in many biological processes such as transcriptional regulation, gene imprinting, X chromosome inactivation, transposon silencing, and embryonic development. With the development of next-generation sequencing technology, a large number of high-throughput methylation data are constantly emerging, and the processing and analysis of these data is an urgent problem to be solved. This review discussed the difficulties and challenges encountered in the analysis of WGBS methylation data from four levels: (i) Cytosines to Reads: Technology based on bisulfite conversion; (ii) Reads to Methylation level: BS-seq Sequence alignment; (iii) Methylation level to Region: Characteristics of the methylation group; (iv) Muticle methylomes: Differential methylation. In particular, we discussed the effects of cellular heterogeneity, other cytosine modifications at the site, region, and multiple methylation levels on WGBS methylation data.