Q-RRBS: a quantitative reduced representation bisulfite sequencing method for single-cell methylome analyses

Abstract

Reduced representation bisulfite sequencing (RRBS) is a powerful method of DNA methylome profiling that can be applied to single cells. However, no previous report has described how PCR-based duplication-induced artifacts affect the accuracy of this method when measuring DNA methylation levels. For quantifying the effects of duplication-induced artifacts on methylome profiling when using ultra-trace amounts of starting material, we developed a novel method, namely quantitative RRBS (Q-RRBS), in which PCR-induced duplication is excluded through the use of unique molecular identifiers (UMIs). By performing Q-RRBS on varying amounts of starting material, we determined that duplication-induced artifacts were more severe when small quantities of the starting material were used. However, through using the UMIs, we successfully eliminated these artifacts. In addition, Q-RRBS could accurately detect allele-specific methylation in absence of allele-specific genetic variants. Our results demonstrate that Q-RRBS is an optimal strategy for DNA methylation profiling of single cells or samples containing ultra-trace amounts of cells.

Acetylation of hMOF Modulates H4K16ac to Regulate DNA Repair Genes in Response to Oxidative Stress

“…To examine whether DNA methylation affects the transcriptional alteration of DNA repair genes, we performed reduced representation bisulfite sequencing (RRBS-seq) in HCT116 cells after H 2 O 2 treatment. We identified 785 differentially methylated regions (DMRs) using a sliding window approach 32 . The differential methylation levels in H 2 O 2 -treated HCT116 were observed to occur on genomic features of 3' UTR, 5' UTR, CDS, intron, and promoter regions (Fig.…”

Base-resolution profiling of active DNA demethylation using MAB-seq and caMAB-seq

“…Second, RRBS preferentially cover GC-rich regions that are generally depleted of 5fC/5caC (at least in mouse ES cells). These potential problems can be in part solved by adopting unique molecule identifier (UMI)-based RRBS approach 66 to remove PCR duplicates, and by using other restriction enzymes that cover more 5fC/5caC-modified regions.…”

Advances in the Profiling of Single‐Cell DNA Modifications

“…However, considering the “reduced representation” from restriction digestion, degradation from bisulfite treatment and PCR amplification bias, scRRBS has only ≈4% coverage of the CpG sites, compared with ≈10% coverage detected by bulk RRBS . There is another similar method called quantitative RRBS (Q‐RRBS) developed to detect allele‐specific methylation of single cells through introducing unique molecular index (UMI) which can track duplication‐induced artifacts …”