RESEARCH ARTICLE Random Tagging Genotyping by Sequencing (rtGBS), an Unbiased Approach to Locate Restriction Enzyme Sites across the Target

Genotyping by sequencing (GBS) is a restriction enzyme based targeted approach devel-oped to reduce the genome complexity and discover genetic markers when a priori sequence information is unavailable. Sufficient coverage at each locus is essential to distinguish hetero-zygous from homozygous sites accurately. The number of GBS samples able to be pooled in one sequencing lane is limited by the number of restriction sites present in the genome and the read depth required at each site per sample for accurate calling of single-nucleotide poly-morphisms. Loci bias was observed using a slight modification of the Elshire et al. method: some restriction enzyme sites were represented in higher proportions while others were poorly represented or absent. This bias could be due to the quality of genomic DNA, the endonuclease and ligase reaction efficiency, the distance between restriction sites, the pref-erential amplification of small library restriction fragments, or bias towards cluster formation of small amplicons during the sequencing process. To overcome these issues, we have developed a GBSmethod based on randomly tagging genomic DNA (rtGBS). By randomly landing on the genome, we can, with less bias, find restriction sites that are far apart, an