title: UCE target enrichment of Alepisaurus_ferox_YPM_25657 muscle tissue
design: We extracted DNA from 120 species following the DNeasy protocol for extraction (Qiagen, Inc.). Following elution, we quantified 2 ľL of DNA using a Qubit fluorometer (Invitrogen, Inc.) following the manufacturer's protocol. We visualized 50-100 ng of each DNA by electrophoresis through 1.5% (w/v) agarose gel in TBE. We prepared 100 ľL aliquots (~10 ng/ľL) of each sample for sonication, and we sheared each sample to 300-600 bp in length using 5-10 cycles of sonication (High; 30 s on; 90 s off) on a BioRuptor (Diagenode, Inc.).We included two samples (Antennarius striatus, Taenianotus triacanthus) previously prepared, enriched and sequenced as described in (Faircloth et al. 2013). Because we prepared and enriched the remaining 118 samples during a two year period 2012-2014, we used slightly different library preparation and enrichment procedures for particular samples (Table S1). In the case of library preparation, we prepared either single- (Faircloth and Glenn 2012) or dual-indexed (Glenn et al. 2016) sequencing libraries following protocols derived from the general approach described in (Faircloth et al. 2012) and (Faircloth et al. 2013).For preparation of single-indexed samples, we input sheared DNA to a commercial library preparation kit (Kapa Biosystems), we used a generic SPRI substitute (Rohland and Reich 2012) for cleaning steps, we performed full SPRI washing procedures using 70% EtOH between each of the library preparation steps, and we attached single-indexed adapters (Faircloth and Glenn 2012) to DNA from each tissue. Following adapter ligation, we amplified 50% of the library volume (~15 uL) using a reaction mix of 25 ľL HiFi HotStart ReadyMix polymerase (Kapa Biosystems), 5 ľL of Illumina P5 and P7 primer mix (5 ľM each), and 5 ľL double-distilled water (ddH20) using the following thermal protocol: 98 ĄC for 45s; 14-16 cycles of 98 ĄC for 15s, 60 ĄC for 30s, 72 ĄC for 60s; and a final extension of 72 ĄC for 5m.For preparation of double-indexed samples, we input sheared DNA to a Ówith-bead'' (Fisher et al. 2011) library preparation process using a commercial library preparation kit (Kapa Biosystems) and a generic SPRI substitute (Rohland and Reich 2012). During adapter ligation, we substituted a custom-designed set of adapters that allows dual-indexing during the subsequent PCR amplification phase (Glenn et al. 2016). Following universal adapter ligation, we amplified 50% of the library volume (~15 uL) using a reaction mix of 25 ľL HiFi HotStart ReadyMix polymerase (Kapa Biosystems), 5 ľL of dual-indexed primer mix (5 ľM each), and 5 ľL double-distilled water (ddH20) using the following thermal protocol: 98 ĄC for 45s; 10-12 cycles of 98 ĄC for 15s, 60 ĄC for 30s, 72 ĄC for 60s; and a final extension of 72 ĄC for 5m. For both library preparation procedures, we purified all resulting PCR products using 1.8X SPRI, and we rehydrated libraries in 33 ľL ddH2O or 10 mM Tris-HCl. We quantified 2 ľL of each library using a Qubit fluorometer, and we combined 62.5 ng DNA from each sample into pools of eight libraries at equimolar ratios (500 ng each pool). The final concentration of each pool was 147 ng/ľL.We enriched UCE loci from pooled sequencing libraries following a standardized protocol (v1.4 and v.1.5 available from http://ultraconserved.org) modified from (Blumenstiel et al. 2010)using 100 ng MYcroarray MYBaits baits synthesized from the target enrichment array designed for acanthomorphs. We PCR-recovered enriched libraries for UCE targets following two different protocols that can generally be described as Ňwithout-beadÓ (v1.4) or Ňwith-beadÓ (v1.5) approaches. For the Ňwithout-beadÓ approach, we dissociated enriched DNA from RNA baits bound to streptavidin-coated beads by combining beads with 0.1N NaOH (Blumenstiel et al. 2010) for 10 m, followed by a five minute neutralization of NaOH using an equal volume of 1 M Tris-HCl. We cleaned the neutralized enrichments using a 1X SPRI cleanup followed by elution of the SPRI-purified sample in 30 ľL of ddH2O or 10 mM Tris-HCL. We combined 15 ľL of the enriched library in water with 25 ľL HiFi HotStart ReadyMix polymerase (Kapa Biosystems), 5 ľL of Illumina TruSeq primer mix (5 ľM each), and 5 ľL of ddH2O. For the Ňwith-beadÓ approach, we removed the final aliquot of wash buffer and allowed samples to dry for five minutes while sitting in a magnet stand. We removed residual buffer with sterile toothpicks. Then, we added 30 ľL ddH20 to each sample, mixed each sample, and proceeded directly to PCR recovery while the enriched libraries were still bound to streptavidin beads (Fisher et al. 2011; Faircloth et al. 2015). We combined 15 ľL of the streptavidin bead-bound, enriched library in water with 25 ľL HiFi HotStart ReadyMix polymerase (Kapa Biosystems), 5 ľL of Illumina TruSeq primer mix (5 ľM each), and 5 ľL of ddH2O.We performed PCR recovery of both post-enrichment treatments using the following thermal profile: 98 ĄC for 45s; 16 cycles of 98 ĄC for 15s, 60 ĄC for 30s, 72 ĄC for 60s; and a final extension of 72 ĄC for 5m. We purified resulting reactions using 1.0X SPRI, and we re-hydrated enriched pools in 33 ľL ddH2O. We quantified 2 ľL of each enriched pool using a Qubit fluorometer, and we diluted PCR-recovered, enriched pools to 2.5 ng/ľL. We determined the fragment size distribution of each enriched pool using a Bioanalyzer (Agilent Technologies, Inc.), qPCR-quantified each pool using a commercial library quantification kit (Kapa Biosystems), and adjusted estimated pooled library concentrations based on our qPCR results and the mean fragment size of each pool. We created a equimolar pools-of-all-pools at 10 nM concentration for sequencing, and we sequenced 10 nM pools-of-pooled libraries over several runs of Illumina sequencing on either the MiSeq or the HiSeq platform with different target read lengths (Table S1).