The use of the flanking DNA sequences around each restriction site is an important aspect of RAD tags. The density of RAD tags in a genome depends on the restriction enzyme used during the isolation process. There are other restriction site marker techniques, like RFLP or amplified fragment length polymorphism, which use fragment length polymorphism caused by different restriction sites, for the distinction of genetic polymorphism. The use of the flanking DNA-sequences in RAD tag techniques is referred as reduced-representation method. The initial procedure to isolate RAD tags involved digesting DNA with a particular restriction enzyme, ligating biotinylated adapters to the overhangs, randomly shearing the DNA into fragments much smaller than the average distance between restriction sites, and isolating the biotinylated fragments using streptavidin beads. This procedure was used initially to isolate RAD tags for microarray analysis. More recently, the RAD tag isolation procedure has been modified for use with high-throughput sequencing on the Illuminaplatform, which has the benefit of greatly reduced raw error rates and high throughput. The new procedure involves digesting DNA with a particular restriction enzyme, ligating the first adapter, called P1, to the overhangs, randomly shearing the DNA into fragments much smaller than the average distance between restriction sites, preparing the sheared ends into blunt ends and ligating the second adapter, and using PCR to specifically amplify fragments that contain both adapters. Importantly, the first adapter contains a short DNA sequence barcode, called MID that is used as a marker to identify different DNA samples that are pooled together and sequenced in the same reaction. The use of high-throughput sequencing to analyze RAD tags can be classified as reduced-representation sequencing, which includes, among other things, RADSeq.
Detection and genotyping of RAD markers
Once RAD tags have been isolated, they can be used to identify and genotype DNA sequence polymorphisms such as single nucleotide polymorphisms. These polymorphic sites are referred to as RAD markers. The most efficient way to find RAD tags is by high-throughput DNA sequencing, called RAD tag sequencing, RAD sequencing, RAD-Seq, or RADSeq. Prior to the development of high-throughput sequencing technologies, RAD markers were identified by hybridizing RAD tags to microarrays. Due to the low sensitivity of microarrays, this approach can only detect either DNA sequence polymorphisms that disrupt restriction sites and lead to the absence of RAD tags or substantial DNA sequence polymorphisms that disrupt RAD tag hybridization. Therefore, the genetic marker density that can be achieved with microarrays is much lower than what is possible with high-throughput DNA-sequencing.
History
RAD markers were first implemented using microarrays and later adapted for NGS. It was developed jointly by Eric Johnson and William Cresko's laboratories at the University of Oregon around 2006. They confirmed the utility of RAD markers by identifying recombination breakpoints in D. melanogaster and by detecting QTLs in threespine sticklebacks. In 2012, scientists published a modified RAD tagging method called double digest RADseq. They added a second restriction enzyme and a tight DNA size selection step to perform low-cost population genotyping.
