RAD-Seq – A brief technical overview
Some time ago I was introducing a new approach combining restriction site associated DNA marker genotyping (RAD) with next generation sequencing technology. Originally this method was developed for microarray platforms. However, the combination of RAD and NGS (Illumina) – resulting in RAD sequencing (RAD-Seq) – enabled the massivly parallel and multiplexed sample sequencing. RAD-Seq is becoming more and more powerful and has the potential to revolutionize agrigenomics, because one can discover and screen thousands of SNP’s and genotype large populations in a high throughput manner at the same time. The scope of the following section is to give a short technical overview how this can be accomplished:
Genomic DNA of each sample is digested in parallel with a certain restriction enzyme and a specific P1 adapter is ligated to the restriction fragments. Thereby each sample will be equipped with an individual P1-adapter containing a sample-specific molecular identifier (Barcode) and Illumina adapter sequences (forward amplification primer site and Illumina sequencing primer site, respectively). If multiplexing is desired, the adapter-ligated fragments of a number of samples can now be pooled. The level of multiplexing depends on the number of differed P1-adapters which have been used before. In a further step the RAD pool will be sheared, size-selected and ligated with a second adapter (P2). The P2 adapter comprises a divergent “Y” adapter containing the reverse amplification primer sites. However, the P2 adapter is special such that fragments lacking the P1 adapter cannot be amplified. This guarantees, that only fragments containing a P1 and a P2 adapter will be selectively and robustly enriched during amplification step following next. The overall length of RAD-tags which can be further analysed mainly depend on the size selection step and sequencing run mode (single vs. paired end), respectively.