Gel-free multiplexed reduced representation bisulfite sequencing for large-scale DNA methylation profiling

DNA methylation plays an important role in mammalian development [1,2] and is frequently altered in diseases, including cancer [3]. It is generally thought that methylation acts in a repressive function within regulatory contexts [4,5]. DNA methylation in mammalian genomes occurs mostly within the context of the CpG dinucleotide [6] and is generally seen in CpG-poor regions. In contrast, CpG-rich regions naturally exhibit low methylation states [7-10].Many techniques have been developed to investigate global DNA methylation patterns [11]. Comparison of next-generation sequencing-based technologies showed that most methods produce similar results [12,13], but that the optimal sequencing strategy may depend on sample DNA amount, as well as the desired genome coverage and sequencing depth [14,15]. Whole-genome bisulfite sequencing of randomly sheared genomic DNA is the most comprehensive, but also most costly, method, while more focused approaches such as reduced representation bisulfite sequencing (RRBS) allow larger numbers of samples to be analyzed at reduced costs [8,15-17].RRBS utilizes the cutting pattern of MspI (C^CGG) to systematically digest DNA to enrich for CpG dinucleotides. As opposed to whole-genome bisulfite sequencing, every fragment produced by MspI digestion will contain DNA methylation information for at least one CpG dinucleotide [6]. Another benefit of RRBS is that promoters, CpG islands, and other genomic features are disproportionally enriched genomic features because of the frequency of MspI cut sites in these regions [8,16].RRBS reduces the complexity of the genome - and thus the sequencing cost - by selecting a subset of MspI fragments based on their size for sequencing. In the standard RRBS protocol, this size selection is done by preparative gel electrophoresis, which is laborious and difficult to automate, thereby limiting the throughput of the method. For example, using our more recently published protocol [15], which includes a manual 40