title: GBS of Acacia koaia: leaf tissue
design: Total genomic DNA was extracted from frozen tissue using the NucleoSpin kit (Macherey-Nagel, Bethlehem, PA). DNA was prepared for sequencing using an efficient restriction-enzyme-based approach commonly known as genotyping by sequencing (GBS) (Elshire et al., 2011 PLoS ONE). Briefly, DNA was digested with the ApeKI restriction enzyme, common and unique barcoded adapters with overhangs complementary to the cut site were ligated to each sample, 48 samples were pooled in equimolar ratios, and the pooled library was PCR-amplified and sent for Illumina sequencing. We largely followed the original protocol of Elshire et al. (2011), including using the same adapter concentration. However, a different set of longer barcode sequences were used (Gugger et al. submitted Evolutionary Applications); all steps were performed manually rather than robotically; adapters were added during the ligation step rather than added to the empty plate and dried down prior to digestion; AMPure XP bead-based size selection/purification steps were added after the ligation step and repeated after the PCR step to ensure a consistent distribution of fragment sizes between 200 and 500 bp among all preps; and we reduced the number PCR cycles to 16 from 18. Libraries were sent at the UCLA Broad Stem Cell Research Center for single-end, 100-bp sequencing on an Illumina HiSeq2000 v3 (San Diego, CA). Resulting sequence data in FASTQ format was quality filtered and demultiplexed using Ôprocess_radtagsŐ from Stacks 1.35 (Catchen et al. 2013) which removed adapter sequence with up to two mismatches (--adapter_mm), recovered reads whose barcodes had up to one mismatch to the expected barcodes (-r), removed any read with an uncalled base (-c), discarded low-quality reads as defined by default settings (-q), and trimmed all reads to 87 bases (-t).