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

• DOI: 10.1371/journal.pone.0143193
• Publication Date: 2015-12-03T18:50:40Z
• Authors: Elena Hilario; Lorna Barron; Cecilia H. Deng; Paul M. Datson; Nihal De Silva; Marcus W. Davy; Roy D. Storey
• Keywords: Restriction site; Amplicon; Genome size

Genotyping by sequencing (GBS) is a restriction enzyme based targeted approach developed to reduce the genome complexity and discover genetic markers when priori sequence information unavailable. Sufficient coverage at each locus essential distinguish heterozygous from homozygous sites accurately. The number of GBS samples able be pooled in one lane limited present read depth required site per sample for accurate calling single-nucleotide polymorphisms. Loci bias was observed using slight modification Elshire et al. method: some were represented higher proportions while others poorly or absent. This could due quality genomic DNA, endonuclease ligase reaction efficiency, distance between sites, preferential amplification small library fragments, towards cluster formation amplicons during process. To overcome these issues, we have method on randomly tagging DNA (rtGBS). By landing genome, can, with less bias, find that are far apart, undetected standard (stdGBS) method. study comprises two types biological replicates: six different kiwifruit plants independent extractions plant; three technical four extraction, stdGBS vs. rtGBS methods, amplifications, sequenced separate lanes. A statistically significant unbiased distribution fragment size showed this 49% (39,145) BamH I shared reference compared only 14% (11,513) stdGBS.