title: GBS of Tricalysia sp.: leaf
design: DNA was extracted using an optimized cetyltrimethylammonium bromide (CTAB) protocol adapted from Doyle & Doyle (1987). GBS libraries were prepared using a single-enzyme protocol slightly adapted from Elshire et al. (2011). First, 100 ng of DNA was digested with PstI (New England Biolabs, Ipswitch, USA). The digested DNA fragments were ligated to a barcode adapter-common adapter system (0.045 pmol) with T4 DNA ligase (New England Biolabs, Ipswitch, USA). Ligation products were purified with 1.6X MagNa magnetic beads (Rohland & Reich, 2012) and eluted in 30 l TE. Of the purified DNA eluate, 3 l were used for amplification with Taq 2X Master Mix (New England Biolabs, Ipswitch, USA) using a 20-cycles PCR protocol. PCR products were bead-purified with 1.6X MagNa, and equimolar amounts of the GBS libraries were pooled, bead-purified, and 150 bp paired-end sequenced on an Illumina HiSeq-X instrument by Admera Health (South Plainfield, USA). Afterwards, reads were demultiplexed using GBSX v1.3 (Herten et al., 2015) with one mismatch allowed in barcodes. The maximum length of forward reads was adjusted to 142 bp in order to compensate for variable barcode lengths. The 3 restriction site remnant and the common adapter sequence of forward reads and the 3 restriction site remnant, the barcode, and the barcode adapter sequence of reverse reads were removed with Cutadapt v1.9 (Martin, 2011). The 5 restriction site remnant of forward and reverse reads was trimmed with FASTX-Toolkit v0.0.13 (Gordon & Hannon, 2010). The trimmed fastq files were submitted to the SRA.