Stacks 2: Analytical methods for paired‐end sequencing improve RADseq‐based population genomics

In the last 20 years, we have observed an exponential growth of the DNA sequence data and simular increase in the volume of DNA polymorphism data generated by numerous molecular marker technologies. Most of the investment, and therefore progress, concentrated on human genome and genomes of selected model species. Diversity Arrays Technology (DArT), developed over a decade ago, was among the first "democratizing" genotyping technologies, as its performance was primarily driven by the level of DNA sequence variation in the species rather than by the level of financial investment. DArT also proved more robust to genome size and ploidy-level differences among approximately 60 organisms for which DArT was developed to date compared to other high-throughput genotyping technologies. The success of DArT in a number of organisms, including a wide range of "orphan crops," can be attributed to the simplicity of underlying concepts: DArT combines genome complexity reduction methods enriching for genic regions with a highly parallel assay readout on a number of "open-access" microarray platforms. The quantitative nature of the assay enabled a number of applications in which allelic frequencies can be estimated from DArT arrays. A typical DArT assay tests for polymorphism tens of thousands of genomic loci with the final number of markers reported (hundreds to thousands) reflecting the level of DNA sequence variation in the tested loci. Detailed DArT methods, protocols, and a range of their application examples as well as DArT's evolution path are presented. Show more

We describe 2b-RAD, a streamlined restriction site-associated DNA (RAD) genotyping method based on sequencing the uniform fragments produced by type IIB restriction endonucleases. Well-studied accessions of Arabidopsis thaliana were genotyped to validate the method's accuracy and to demonstrate fine-tuning of marker density as needed. The simplicity of the 2b-RAD protocol makes it particularly suitable for high-throughput genotyping as required for linkage mapping and profiling genetic variation in natural populations. Show more