UCE target enrichment of Repomucenus belcheri: muscle tissue
We extracted DNA from ethanol-preserved tissues following a modified Qiagen DNeasy protocols. We quantified all DNA extracts with a Qubit Fluorometer, assessed each extract for quality by electrophoresis, and then sheared extracts by sonication to a target size of 600bp on a Bioruptor (Diagenode, Inc.). Library preparation and targeted enrichment for UCEs followed protocols described in detail by McGee et al. (2016, http://dx.doi.org/10.1098/rspb.2015.1413),  which are also available online (http://ultraconserved.org). In brief, we prepared libraries from sheared DNA using commercially available library preparation regents (Kapa Biosystems), a generic SPRI substitute and custom adapters  (Glenn et al., 2016, BioRxiv 049114). Following amplification, we grouped libraries into pools of eight based on taxonomic similarity, and we enriched pooled libraries for UCE targets using Mycoarray MYbaits UCE Capture Kits designed to target approximately 1,340 highly conserved loci across acanthomorph fishes. We determined the size of enriched and purified pools with a Bioanalyzer (Agilent Technologies, Inc.) and we quantified enriched libraries by qPCR (Kapa Biosystems) prior to sequencing. We sequenced libraries using approximately 2/3 of an Illumina NextSeq PE150 run (Georgia Genomics Facility). We demultiplexed and converted reads for downstream processing using bcl2fastq2 conversion software (v02.14.01.07. Illumina Inc.). These are the raw, untrimmed reads.