SRP008452 2b-RAD method 2b-RAD method Sequencing restriction-site associated DNA (RAD) tag libraries is a powerful and cost-effective approach for profiling genetic variation at the whole-genome scale. Here we introduce an improved version called 2b-RAD for its use of type IIB restriction enzymes to produce fragments of uniform length. 2b-RAD analysis of Arabidopsis thaliana samples from resequenced accessions is presented to demonstrate the method’s power and accuracy. We describe reference-based and de novo analytical methods for 2b-RAD genotyping, procedures for customizing library preparation to optimize marker densities, and demonstrate the effectiveness of 2b-RAD in the larger human genome. 2b-RAD provides a flexible and cost-effective method for studying the genetic variation underlying disease, natural phenotypic variation, and evolutionary adaptation. 2b-RAD method Restriction fragments produced using type IIb restriction endonucleases (BsaXI, AlfI) were sequenced using SOLiD or Illumina.