Genotyping-By-Sequencing Library Preparation

Using Elshire et al.’s (2011) (link) methodology with modifications, GBS data were generated. In brief, each 200 ng of DNA sample was digested with five units of the ApeK1 enzyme at 75°C for 4 h and cooled down to 4°C, followed by GBS library preparation. Unique barcodes and a “common” adapter were used for each individual ligation. After ligation, they were pooled and purified using AMpure DNA purification beads and the genomic fragments were then amplified in 50-µl volumes. GBS library purification was carried out using Agencourt AMPure XP SPRI magnetic beads (Beckman Coulter). The quality of the GBS library was assessed using the 4150 TapeStation System (Catalog: G2992AA, Agilent). The GBS library was sequenced using the Illumina HiSeq 2000 platform with paired-end (PE) reads of 150 × 2 bp.