title: ddRADseq Monechma
design: All genomic DNA was normalized to ~30 ng/ml before digestion and library construction. Extracted DNA underwent double restriction enzyme digestion using EcoR1 and MseI for 3 hours at 37 followed by 65 for 45 min. Illumina sequencing oligos together with in-line, variable-length barcodes were annealed to the EcoR1 cut site and ligated onto digested fragments. Illumina oligos were similarly annealed to the MseI cutsite. Barcoded ligation products were pooled and cleaned using a Qiagen gel extraction kit. We excised fragments from the gel between 200-700 bp to reduce the effects of dimer and to provide more precise amplification of the targeted region. The gel-purified ligations were amplified using PCR reactions. Agarose gels were used to assess amplification and size of the PCR products and amplicon concentrations were evaluated using a Qubit fluorometer 2.0. The custom-tagged products of the PCR reactions were pooled and sent to the University of Colorado's Biofrontiers Next-Gen Sequencing Facility for quality control and further size selection. BluePippin was used to select a fragment range between 200 and 500 bp to reduce the sequenced genome. Libraries from the 80 samples were pooled to yield a final combined library that was submitted for 1x75 sequencing on an Illumina NextSeq v2 High Output Sequencer at Biofrontiers.