Targeted Genotyping-By-Sequencing with Single Primer Enrichment Technology (SPET). a Case Study in Black Poplar.

Genotyping-by-sequencing has now become a widely adopted strategy to generate massive genotypic information to a fraction of the cost used in array technologies. Sequencing-based genotyping carries several advantages such as greater scalability, given the current sequencing platforms, and being an “open system”, allowing for the scoring of polymorphisms that were not necessarily a prior knowledge. However, one major limitation of widely adopted genotyping-by-sequencing methods, driven by restriction enzymes or oligomers, is its random access to the genome. On the other side, enrichment methodologies based on hybridization come with some limitation in cost, hand-on time, enrichment efficiency and dilution of the sequencing coverage on valuable sites. Here we propose the NuGEN Ovation Target Enrichment - a method originally developed for exon-centric enrichment - as a unique tool for large-scale targeted genotyping. Slight modification to the original sequencing protocol allowed to generate RAD-like stacks of reads on 90,000 pre-selected SNPs over 540 black poplar genotypes, maximizing efficiency. Moreover, the sequencing of extra bases, from both paired reads, allowed the discovery of hundreds of thousands novel SNPs, sampling rarer variants not originally included in the panel. The system showed its powerful combination of array-like target SNP paradigm along with the random sampling of variants of traditional genotyping-by-sequencing solutions, circumventing ascertainment bias. Data showed complete usability for the discovery of trait-associated polymorphisms within the 7^th Framework Programme EU-funded project WATBIO.