Botaurus_lentiginosus_LSUMNS_B18981_UCE
We standardized our extractions by including 250 ng of DNA in a 50 L aliquots, which were subsequently fragmented with a Covaris S220 focus-ultrasonicator with the following settings: 175 W peak incident power, a duty factor of 2%, and 200 cycles per bust for 44-45 seconds. We targeted fragments of 500-600 bp in length. We then prepared libraries using Kapa Biosystems Library Hyper Prep Kits (# KK2602), following manufacturers protocol, with some minor modifications: 25 l per sample, at a concentration of 10 ng/l, was subjected to end repair and A-tailing on a thermal cycler, followed by the ligation of two universal iTru stubs (iTru Stub Oligo 1 & iTru Stub Oligo 2) and incorporation of iTru dual-indexes (Glenn et al. 2016). Following ligation, we purified samples with Agencourt AMPure XP beads, at a volume of 0.8X. We then amplified libraries using the manufacturers recommended PCR protocol, which included 10 cycles for the denaturation, annealing, and extension steps. We then performed an additional 1X Agencourt AMPure XP bead cleanup, followed by a quantification of the amplified libraries using a Qubit dsDNA HS Assay Kit on a Qubit 2.0 Fluorometer (Life Technologies). Pools of eight amplified libraries were then grouped and enriched for 5,060 UCE loci using 5,742 baits in the MYbaits Tetrapods-UCE-5kv1-96 library capture kit (sequences available at ultraconserved.org) (Faircloth et al. 2012), following the Mycroarray myBaits  3.01 Kit manufacturers protocol. After enrichment, we subsequently amplified capture-reaction products using 18 PCR cycles, followed by a 1.2X Agencourt AMPure XP bead cleanup. Lastly, we quantified enriched, pooled libraries using a Qubit dsDNA HS Assay Kit on a Qubit 2.0 Fluorometer (Life Technologies). Prior to sequencing, pools consisting of equimolar libraries were combined.