RAD With Type IIB Restriction Endonucleases

2b-RAD is similar to ddRadseq but uses type IIB restriction enzymes (BsaXI or AlfI), which will cleave upstream and downstream of a recognition site(Wang et al., 2012) . This shears the target genome into a large number of DNA fragments with a constant length of 33 bp (BsaXI) or 36 bp (AlfI). These short DNA fragments can be sequenced to determine genetic variants.

In this method, gDNA is first digested with a restriction enzyme (BsaXI), and adapters with partial (NNN) overhangs are ligated to the fragments. The adapter-ligated fragments from different samples are combined, and the fragments are amplified to produce the sequencing library.


  • Highly reduced 2b-RAD libraries require much less sequencing for accurate genotyping
  • High density of markers
  • No interim purification steps, reducing losses and processing time


  • Requires a reference genome
  • Short tags may not be long enough for efficient locus discrimination in complex genomes


Illumina Library prep and Array Kit Selector


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