title: 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.
authors.affiliation: a Institute of Genomic Medicine; Wenzhou Medical University ; Wenzhou , China.
authors.forename: Kangli
authors.initials: K
authors.lastname: Wang
authors.forename: Xianfeng
authors.initials: X
authors.lastname: Li
authors.forename: Shanshan
authors.initials: S
authors.lastname: Dong
authors.forename: Jialong
authors.initials: J
authors.lastname: Liang
authors.forename: Fengbiao
authors.initials: F
authors.lastname: Mao
authors.forename: Cheng
authors.initials: C
authors.lastname: Zeng
authors.forename: Honghu
authors.initials: H
authors.lastname: Wu
authors.forename: Jinyu
authors.initials: J
authors.lastname: Wu
authors.forename: Wanshi
authors.initials: W
authors.lastname: Cai
authors.forename: Zhong Sheng
authors.initials: ZS
authors.lastname: Sun
mesh_terms: D000818:Animals; D018899:CpG Islands; D019175:DNA Methylation; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D059010:Single-Cell Analysis / Q000379:methods*
keywords: DNA methylome; Q-RRBS; allele-specific methylation; deduplication; single cell; unique molecular identifiers