DNA methylome profiling at single-base resolution through bisulfite sequencing of 5mC-immunoprecipitated DNA

Abstract Background Detection of DNA methylome at single-base resolution is a significant challenge but promises to shed considerable light on human disease etiology. Current technologies could not detect DNA methylation genome-wide at single-base resolution with small amount of sequencing data and could not avoid detecting the methylation of repetitive elements which are considered as “junk DNA”. Methods In this study, we have developed a novel DNA methylome profiling technology named MB-seq with its ability to identify genome-wide 5mC and quantify DNA methylation levels by introduced an assistant adapter AluI-linker This linker can be ligated to sonicated DNA and then be digested after the bisulfite treatment and amplification, which has no effect of MeDIP enrichment. Because many researchers are interested in investigating the methylation of functional regions such as promoters and gene bodies, we have also developed a novel alternative method named MRB-seq, which can be used to investigate the DNA methylation of functional regions by removing the repeats with Cot-1 DNA. Results In this study, we have developed MB-seq, a novel DNA methylome profiling technology combining MeDIP-seq with bisulfite conversion, which can precisely detect the 5mC sites and determine their DNA methylation level at single-base resolution in a cost-effective way. In addition, we have developed a new alternative method, MRB-seq (MeDIP-repetitive elements removal-bisulfite sequencing), which interrogates 5mCs in functional regions by depleting nearly half of repeat fragments enriched by MeDIP. Comparing MB-seq and MRB-seq to whole-genome BS-seq using the same batch of DNA from YH peripheral blood mononuclear cells. We found that the sequencing data of MB-seq and MRB-seq almost reaches saturation after generating 7–8 Gbp data, whereas BS-seq requires about 100 Gbp data to achieve the same effect. In comparison to MeDIP-seq and BS-seq, MB-seq offers several key advantages, including single-base resolution, discriminating the methylated sites within a CpG and non-CpG pattern and overcoming the false positive of MeDIP-seq due to the non-specific binding of 5-methylcytidine antibody to genomic fragments. Conclusion Our novel developed method MB-seq can accelerate the decoding process of DNA methylation mechanism in human diseases because it requires 7–8 Gbp data to measure human methylome with enough coverage and sequencing depth, affording it a direct and practical application in the study of multiple samples. In addition, we have also provided a novel alternative MRB-seq method, which removes most repetitive sequences and allows researchers to genome-wide characterize DNA methylation of functional regions

Additional file 1: of DNA methylome profiling at single-base resolution through bisulfite sequencing of 5mC-immunoprecipitated DNA

Table S1. No adverse effect of methylated AluI-linker on MeDIP enrichment. Table S2. Evidence of a decreased percentage of sequenced AluI-linker following removal with streptavidin coupled beads. Table S3. AluI-linker sequences and primers used in this study. Table S4. The qPCR primers used to evaluate MeDIP recovery efficiency. Figure S1. Comparison of the concordance mCG sites identified from MB-seq and BS-seq. Figure S2. Example for the false positive exclusion of MeDIP-seq by MB-seq. Figure S3. The differences of read depth of each genomic feature between MB-seq and MRB-seq based on D-value distribution. Figure S4. Scatter plots showing the relationship between density of methyl groups in MB-Seq and MRB-seq for different genomic features. Figure S5. The methylation levels of BS-seq in gene-associated regions. Figure S6. Comparison of the methylation level differences between BS-seq, MB-seq and MRB-seq. Figure S7. Coverage of CGI and none-CGI promoter. (a) Coverage of each CGI distributed with methylation of C on genome. (b) Coverage of each none CGI promoter distributed with methylation of C on genome. (DOC 2899 kb