Multiplexed Reduced Representation Bisulfite Sequencing with Magnetic Bead Fragment Size Selection.

Reduced representation bisulfite sequencing (RRBS) is a technique for assessing genome-wide DNA methylation in an organism whose genome has been fully sequenced. It allows researchers to target gene regions with particular CpG densities, thereby selecting the desired genomic contexts. Here, we describe an approach that uses magnetic beads to accomplish this selection. In addition, the use of indexed, methylated adapters enables up to 12 samples to be pooled, and subjected to multiplexed RRBS in a single-sequencing lane. First, genomic DNA is fragmented via restriction endonuclease digestion that ensures at least two CpG loci per fragment. The fragmented DNA is then end-repaired and A-tailed. Indexed, methylated adapters are ligated to the A-tailed DNA fragments to create a DNA library. A combination of negative and positive selections, using magnetic beads that preferentially bind to larger DNA fragments, ensures that only the desired sizes of adapter-ligated DNA fragments are included in a library. This allows researchers to dictate what types of genomic regions will be sequenced, since fragment size depends on the proximity of restriction sites. The DNA libraries are then quantified, and up to 12 libraries are pooled in order to be sequenced on a single lane of an Illumina HiSeq2500. The pools are next treated with sodium bisulfite, and then PCR amplified. A final bead cleanup removes any residual contaminants prior to sequencing, which is followed by base calling and alignment to a sequenced genome.