Enhanced reduced representation bisulfite sequencing for assessment of DNA methylation at base pair resolution

Journal of Visualized Experiments : JoVE
Francine E Garrett-BakelmanAri Melnick

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 designa...Continue Reading

Citations

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Methods Mentioned

BETA
RRBS
ERRBS
PCR
electrophoresis

Software Mentioned

PhiX
MassARRAY EpiTYPER
SAMTOOLS
R
Bismark
UCSC Genome Browser
IGV
cutadapt
trimmomatic
CASAVA

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