GBS of Quercus lobata: leaf tissue
Total genomic DNA was prepared for sequencing using an efficient restriction-enzyme-based approach commonly known as genotyping by sequencing (GBS) (Elshire et al., 2011). Briefly, DNA was digested with a restriction enzyme, common and unique barcoded adapters with overhangs complementary to the cut site were ligated to each sample, samples were pooled in equimolar ratios, and the pooled library was PCR-amplified and sent for Illumina sequencing. We largely followed the original protocol, including using the same adapter sequences, adapter concentration (0.036 ng/L of each adapter), and restriction enzyme (ApeKI). However, we pooled 48 samples per library prep/sequencing lane rather than 96; adapters were added during the ligation step rather than prior to restriction 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 (including adapters) among all preps; and we reduced the number PCR cycles to 16 from 18. Final libraries were checked for the proper size distribution on an Agilent BioAnalyzer with the High Sensitivity DNA assay and quantified using a Qubit fluorometer. Libraries were sent to the UCLA Broad Stem Cell Research Center for single-end, 100-bp sequencing on an Illumina HiSeq2000 v3. Resulting sequence data in FASTQ format was quality-filtered and demultiplexed using process_radtags 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 92 bases (-t).