Double Digest Restriction-Site Associated DNA Marker Generation

ddRADseq (Peterson et al., 2012), also called ddRAD, is a variation on the RAD sequencing protocol (Baird et al., 2008), which is used for SNP discovery and genotyping (Baird et al., 2008) (Willing et al., 2011). In this variation, the fragment shearing is replaced with a second restriction digestion to improve the tunability and accuracy of the size-selection step. The protocol also includes a second index to allow combinatorial indexing. Several RAD variations, such as 2b-RAD (Wang et al., 2012), SLAF-seq (Sun et al., 2013), and hyRAD (Suchan et al., 2016), have been developed to address specific applications, and multiple software packages are available to analyze RAD data (Fan et al., 2016) (Catchen et al., 2013).

In this method, gDNA is first digested with a restriction enzyme, and a barcoded P1 adapter is ligated to the fragments. The adapter-ligated fragments from different samples are combined, if samples are multiplexed, and the DNA is digested by a second restriction enzyme. The fragments are size-selected and purified. The P2 adapter-primers are ligated, and the fragments are amplified to produce the sequencing library.


  • No reference genome required (Reitzel et al., 2013)
  • Relatively inexpensive, compared to whole-genome sequencing
  • The degree of genome coverage can be adjusted by selecting various restriction enzymes


  • There can be gaps in the genome coverage
  • Requires high-quality DNA (see hyRAD for low-quality DNA) (Suchan et al., 2016)


Illumina Library prep and Array Kit Selector


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