Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

Abstract

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Epigenetic Identity in AML Depends on Disruption of Nonpromoter Regulatory Elements and Is Affected by Antagonistic Effects of Mutations in Epigenetic Modifiers

“…ERRBS was performed as previously described (22, 33). Briefly, DNA was isolated using standard phenol-chloroform extraction and digested with Msp I, followed by end repair, A-tailing, and ligation of methylated Illumina adapters.…”

Neuronal DNA Methylation Profiling of Blast-Related Traumatic Brain Injury

“…36,37 This is a modification of the reduced representation bisulfite sequencing approach, which generates singlebase resolution DNA methylation bisulfite sequencing libraries that enrich for CpG-dense regions by methylation-insensitive restriction digestion. 38 ERRBS is similar to the RRBS method, except longer library fragment lengths of 150-250 bp and 250-400 bp were gel isolated.…”

DNMT3A mutations promote anthracycline resistance in acute myeloid leukemia via impaired nucleosome remodeling

“…The modest DNA hypomethylation relative to other AML alleles with altered DNA methylation 31 as assessed by enhanced reduced representation bisulfite sequencing (ERRBS) 32,33 , the paucity of genes with expression changes correlating with changes in DNA methylation, and the lack of enrichment of pathways relevant to cell survival or drug metabolism in mouse Dnmt3a mut LSK cells compared to wild-type control, suggest that alterations in DNA methylation at specific loci are unlikely to explain anthracycline resistance in DNMT3A R882 mutant cells (Supplementary Fig. 3F–H).…”