individual-GBS of Coffea canephora: leaf
20 - 30 mg of silica-dried leaf material was used to extract DNA of this sample using an optimized cetyltrimethylammonium bromide (CTAB) protocol adapted from Doyle & Doyle (1987). The GBS library was prepared using a double-enzyme protocol that was slightly adapted from Elshire et al. (2011). 100 ng of DNA was digested using PstI and MseI (New England Biolabs, Ipswitch, USA) and ligated to a barcode and common adapter (0.045 pmol) with T4 DNA ligase (New England Biolabs, Ipswitch, USA). Barcode specifications were set as in Verwimp et al. (2018). Ligation products were purified with 1.6X MagNa magnetic beads (Rohland & Reich, 2012) and eluted in 30 l of 0.1X TE. A PCR was performed using 3 l of template DNA, a Taq 2X Master Mix (New England Biolabs, Ipswitch, USA), a unique Illumina indexed i5 barcode for each PCR-replicate, and a 20-cycles PCR program. PCR samples were bead-purified and DNA concentrations were measured with a Quantus Fluorometer. DNA fragment size distributions were evaluated using a Qiagen QIAxcel system (Qiagen, Venlo, NL), and equimolar quantities of each GBS library were pooled, bead-purified, send subject to 150 bp paired-end sequencing on an Illumina HiSeq-X instrument. Afterwards, reads were demultiplexed based on their internal barcode using GBprocesS (Schaumont et al., 2020) with zero mismatch allowed in barcodes. The 3 restriction site remnant, 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 based on sequence-specific pattern recognition and positional trimming using Cutadapt v2.10 (Martin, 2011). The 5 restriction site remnant of forward and reverse reads were trimmed using positional trimming in Cutadapt. Forward reads of each separate individual can be found in *_1.fastq file and reversed reads in *_2.fastq file. Forward and reverse reads with a minimum read length of 60 bp and a minimum overlap of 10 bp were merged using PEAR 0.9.11 (Zhang et al. 2014) and can be found in *_3.fastq file.