Restriction-Site Associated DNA Sequencing

RAD-seq is a protocol for genotyping and discovery of single-nucleotide polymorphisms (SNPs) (Baird et al., 2008). This approach is particularly useful for genotyping when a reference genome is not available, such as in ecological studies (Andrews et al., 2016). PE RAD-seq, also called RAD-PE, is the same protocol as RAD but uses paired-end sequencing for improved alignments (Willing et al., 2011). Several variations, such as ddRADseq (Peterson et al., 2012), 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, genomic DNA (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 sheared. 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 and fragment sizes


  • There can be gaps in the genome coverage
  • Requires high-quality DNA (see hyRAD for low-quality DNA) (Suchan et al., 2016)
  • Sequence polymorphism at the DNA restriction sites causes a progressive loss of shared restriction sites among diverging clades (Suchan et al., 2016)


Illumina Library prep and Array Kit Selector


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