title: Aythya affinis ddRAD-seq library: Sample KGM-677
design: double-digest RAD-seq. Sample preparation for ddRAD sequencing followed the double-digest protocol outlined in DaCosta and Sorenson (2014). In short, ~1 ug of genomic DNA was digested using 10 U of each SbfI and EcoRI restriction enzymes. Adapters containing sequences compatible for Illumina TruSeq reagents and barcodes for de-multiplexing reads were ligated to the sticky ends generated by the restriction enzymes. The adapter-ligated DNA fragments were then size-selected using gel electrophoresis (2% low-melt agarose) and a MinElute Gel Extraction Kit (Qiagen, Valencia, CA). Fragments of 300-450 bp (including adapters) were selected, but fragments as small as ~155 bp are also consistently captured using this method (see DaCosta & Sorenson 2014). Size-selected fragments were then PCR amplified with Phusion high-fidelity DNA polymerase (Thermo Scientific, Pittsburgh, PA), and the amplified products were cleaned using magnetic AMPure XP beads (Beckman Coulter, Inc., Indianapolis, IN). Quantitative PCR using an Illumina library quantification kit (KAPA Biosystems, Wilmington, MA) was used to quantify the concentration of purified PCR products, and samples with compatible barcode combinations were pooled in equimolar concentrations. A multiplexed library was sequenced as a single-end 150 base-pair run on an Illumina HiSeq 2500 at the Tufts University Core Genomics Facility. 