Reduced representation bisulfite sequencing

Reduced representation bisulfite sequencing (RRBS) is an efficient and high-throughput technique for analyzing the genome-wide methylation profiles on a single nucleotide level.

It combines restriction enzymes and bisulfite sequencing to enrich for areas of the genome with a high CpG content.

MspI digestion, or any restriction enzyme that recognizes CpG's and cuts them, produces only fragments with CG’s at the end.

[4] During the PCR portion of the protocol, a non-proofreading polymerase must be used as a proof-reading enzyme would stop at uracil residues found in the ssDNA template.

Complete bisulfite conversion requires thorough denaturation and absence of re-annealed double stranded DNA (dsDNA).

[8] RRBS eliminates the problems encountered due to these large areas of repeated sequences and thus lets these regions be more fully annotated.

[2] This technique can rapidly determine the overall methylation status of cancer genomes which is cost and time effective.

[7] The data obtained on RRBS and the Illumina Infinium methylation are highly comparable, with a Pearson correlation of 0.92.

Finally, Anchor-Based Bisulfite Sequencing (ABBS) was developed by Ben Delatte's group at Active Motif.

This technology uses specialized primers that capture DNA methylation allowing for increased coverage (approx.