title: pseudotropheus-flavus-ID19
design: We extracted DNA from specimen tissues stored in ethanol by cutting 10-20 mg chunks from each tissue using individually wrapped razor blades and following the Qiagen DNeasy protocol.  We modified the protocol by eluting DNA from the column membrane using 65 uL of warm (50-55 °C) Buffer AE.  Following extraction and quantification, we determined the quality of DNA extracts by visualizing 50-100 ng of each extract by electrophoresis through 1.5% (w/v) agarose gel in TBE, prepared 100 µL aliquots (10 ng/µL) 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 prepared sequencing libraries following modified versions of the library preparation protocol v1.10 (available from http://ultraconserved.org). Briefly, we input sheared DNA to a "with-bead" library preparation process using commercial library preparation reagents (Kapa Biosystems) and a generic SPRI substitute ((Rohland & Reich 2012); hereafter SPRI).  During adapter ligation, we substituted a custom-designed set of adapters that allows dual-indexing during the subsequent PCR amplification phase.  Following universal adapter ligation, we amplified 50% of the library volume (~15 uL) using a reaction mix of 25 µL HiFi HotStart ReadyMix polymerase (Kapa Biosystems), 5 µL of dual-indexed primer mix (5 µM each), and 5 µL double-distilled water (ddH20) using the following thermal protocol: 98 C for 45s; 10-12 cycles of 98 C for 15s, 60 C for 30s, 72 C for 60s; and a final extension of 72 C for 5m. We purified resulting reactions using 1.8X SPRI and rehydrated libraries in 33 µL ddH2O.  Prior to targeted enrichment, we selected libraries for grouping based on taxonomic similarity, 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/µL.    We enriched libraries for UCE targets following the enrichment protocol (v. 1.5) available from http://ultraconserved.org and the following reagents: (1) 100 ng MYcroarray MYBaits UCE Capture Kit baits; (2) 500 ng blocking oligos designed against our custom, dual sequence indexes; (3) 500 ng of commercially available chicken Cot-1 DNA (Applied Genetics Laboratories, Inc.); and (4) 1% SDS (versus 10% SDS). We ran the hybridization reaction for 24 hours at 65 C. Following hybridization we bound all pools to streptavidin-coated beads (MyOne C1, Life Technologies, Inc.) and washed bound libraries according to the protocol. Following enrichment, 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, added 30 µL ddH20 to each sample, and then proceeded directly to PCR recovery while the enriched libraries were still bound to streptavidin beads. We combined 15 µL of streptavidin bead-bound, enriched library in water with 25 µL HiFi HotStart ReadyMix polymerase (Kapa Biosystems), 5 µL of Illumina TruSeq primer mix (5 µM each), and 5 µL of ddH2O. We performed PCR recovery of each library using the following thermal profile: 98 C for 45s; 16 cycles of 98 C for 15s, 60 C for 30s, 72 C for 60s; and a final extension of 72 C for 5m. We purified resulting reactions using 1.0X SPRI, and we re-hydrated enriched pools in 33 µL ddH2O.    Prior to sequencing, we diluted PCR-recovered, enriched pools to 2.5 ng/µL, 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 library concentrations based on our qPCR results and the mean fragment size of each pool. We created an equimolar sample of all pools at 10 nM concentration for sequencing, and we sequenced the 10 nM sample of pooled libraries using approximately 1/3 of an Illumina NextSeq PE150 run (Georgia Genomics Facility).