Figure 4

Schematic outline of a typical “Restriction Enzyme Anchored Sequencing” (REAS) approach. (1) DNA from different genotypes is digested with one or several restriction enzymes. (2) Biotinylated adapters that carry a barcode for distinguishing the different genotypes are ligated to the restriction site. (3) A second fragmentation step can be applied, either involving random shearing, or further digestion of the sample with a second restriction enzyme. Biotinylated fragments are then bound to a streptavidin matrix. A second adapter is ligated to the construct that is now ready for sequencing, usually after some PCR cycles. A size selection-step can be included after the first or the second restriction digestion to further reduce the complexity. A mix of both adapters or Y-adapters can also be ligated directly after the first or second digestion or after random shearing. (4) Samples are sequenced and (5) sorted according to barcode, and the tag-sequences are compared for single nucleotide polymorphism detection.