title: Random Tagging Genotyping by Sequencing (rtGBS), an Unbiased Approach to Locate Restriction Enzyme Sites across the Target Genome.
abstract: Genotyping by sequencing (GBS) is a restriction enzyme based targeted approach developed 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 heterozygous 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 polymorphisms. Loci bias was observed using a slight modification of the Elshire et al.
  
  
  METHODS
  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 preferential 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 GBS method 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, and undetected by the standard GBS (stdGBS) method. The study comprises two types of biological replicates: six different kiwifruit plants and two independent DNA extractions per plant; and three types of technical replicates: four samples of each DNA extraction, stdGBS vs. rtGBS methods, and two independent library amplifications, each sequenced in separate lanes. A statistically significant unbiased distribution of restriction fragment size by rtGBS showed that this method targeted 49% (39,145) of BamH I sites shared with the reference genome, compared to only 14% (11,513) by stdGBS.
authors.affiliation: The New Zealand Institute for Plant & Food Research Ltd, Priv. Bag 92-169, Auckland, 1025, New Zealand.
authors.forename: Elena
authors.initials: E
authors.lastname: Hilario
authors.affiliation: The New Zealand Institute for Plant & Food Research Ltd, Priv. Bag 92-169, Auckland, 1025, New Zealand.
authors.forename: Lorna
authors.initials: L
authors.lastname: Barron
authors.affiliation: The New Zealand Institute for Plant & Food Research Ltd, Priv. Bag 92-169, Auckland, 1025, New Zealand.
authors.forename: Cecilia H
authors.initials: CH
authors.lastname: Deng
authors.affiliation: The New Zealand Institute for Plant & Food Research Ltd, Priv. Bag 92-169, Auckland, 1025, New Zealand.
authors.forename: Paul M
authors.initials: PM
authors.lastname: Datson
authors.affiliation: The New Zealand Institute for Plant & Food Research Ltd, Priv. Bag 92-169, Auckland, 1025, New Zealand.
authors.forename: Nihal
authors.initials: N
authors.lastname: De Silva
authors.affiliation: The New Zealand Institute for Plant & Food Research Ltd, 412 No 1 Road RD 2, Te Puke, 3182, New Zealand.
authors.forename: Marcus W
authors.initials: MW
authors.lastname: Davy
authors.affiliation: The New Zealand Institute for Plant & Food Research Ltd, 412 No 1 Road RD 2, Te Puke, 3182, New Zealand.
authors.forename: Roy D
authors.initials: RD
authors.lastname: Storey
mesh_terms: D029042:Actinidia / Q000235:genetics; D004262:DNA Restriction Enzymes; D056426:Genetic Loci; D005819:Genetic Markers; D018745:Genome, Plant; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D020641:Polymorphism, Single Nucleotide
chemical_list: D005819:Genetic Markers; D004262:DNA Restriction Enzymes