Nextseq high output 75 bp single end run

Genotyping-by-sequencing was conducted for 149 enset samples (125 domestic and 24 wild; Supplementary Table 2) that were selected to capture the genetic diversity shown by AFLP.
The GBS library preparation was carried out as described by Xie et al. (2017) including a water negative control as described by Konate et al. (2018) . The DNA concentration of each individual library was normalized to 5 ng/µl. Two pooled libraries were created, each by pooling the individual libraries from 75 uniquely barcoded samples (25 ng per sample) (Supplementary Table 2). Each pooled library was then amplified in 10 PCR reactions, each containing 10 µl of digested/ligated DNA library, 12.5 µl of NEB MasterMix, 2 µl of 10 µM forward and reverse Illumina_PE primers (Supplementary Table 4) and 0.5 µl of molecular biology grade water (Sigma). The amplification reaction was carried using a T1000 Thermocycler at 95 o C for 30 s, 16 cycles of (95 o C for 30 s, 62 o C for 20 s, 68 o C for 30 s) and 72 o C for 5 min. Amplification products were pooled together and cleaned using AMPure XP beads (Beckman Coulter, Australia) (1:1 ratio) to remove excess primers and unremoved adaptors. Libraries were sequenced using an Illumina NextSeq High Output 75 bp single-end run (Illumina 1.9 Inc., San Diego, CA, United States) at the Australian Genome Research Facility (AGRF, Adelaide, SA, Australia).