The Ion PGM Sequencer and a Significantly Streamlined Compatible "Double Digest Restriction Associated Digest"-(ddRAD) Tag Protocol, as a Versatile Probe for Populations, Genetics and Genomics

The Ion Torrent DNA sequencing platform is a versatile and scalable experimental system and can produce read lengths >400 bases using semiconductor technology. To enable genome wide profiling analyses on this platform, using reduce complexity reduction, we developed a double digest Restriction Associated Digest (ddRAD) protocol that is significantly streamlined and compatible with the Ion Torrent platform. Our protocol requires a single combined restriction digestion-adaptor ligation reaction, a fragment size selection and a small pre-amplification step. This system is tunable, particularly in relation to the numbers of markers obtained and the number of individuals profiled, allowing us to appropriately scale our analysis. This is important because we can leverage the differences in size of the semiconductor sensor chips to suit experimental criteria, including depth of coverage and the numbers of markers per individual. A combinatorial barcode system permits the flexibility that several hundred individuals be profiled within several hours of library preparation and sequencing run time. Our test analyses on the Arabidopsis genome do indeed show genome wide coverage and tunability, and we present data on non-model organisms such as Picea abies L. Karst. (1C= >21 Gb), and other plant genomes which are challenging and differ in ploidy level. These results are examined in the context of the power, efficiency and broad range of possibilities that RAD tags and the Ion Torrent system offer.