UCE sequences of Xylocopa_samarensis_232
DNA was extracted destructively from legs of bees using a DNeasy Blood and TissueKit (Qiagen, Valencia, CA, USA). We quantified DNA for each sample using a Qubit fluorometer (High sensitivity kit, Life Technologies, Inc.) and sheared 5.1Đ316 ng DNA for 0Đ60 secs (amp=25, pulse=10) to a target size of approximately 500Đ600 bp by sonication (Q800 or Diagenode BioRuptor; Qsonica Inc.). The sheared DNA was used as input for a modified genomic DNA library preparation protocol (Kapa Hyper Prep Library Kit, Kapa Biosystems) that incorporated Ňwith-beadÓ cleanup steps (Fisher et al. 2011) and a generic SPRI substitute (Rohland and Reich 2012, ŇspeedbeadsÓ hereafter), as described by (Faircloth et al. 2015). We used TruSeq-style adapters during adapter ligation (Faircloth and Glenn 2012), and PCR amplified 50% of the resulting library volume (15 ľL) using a reaction mix of 25 ľL HiFi HotStart polymerase (Kapa Biosystems), 2.5 ľL each of Illumina TruSeq-style i5 and i7 primers (5 ľM each) and 5 ľL double-distilled water (ddH20). We used the following thermal protocol: 98 źC for 45 s; 13 cycles of 98 źC for 15 s, 65 źC for 30 s, 72 źC for 60 s, and final extension at 72 źC for 5 m. After rehydrating (in 23ľL pH 8 Elution Buffer (EB hereafter)) and purifying reactions using 1.0X speedbeads, we combined groups of eight libraries at equimolar ratios into enrichment pools having final concentrations of 107Đ190 ng/ľL. We enriched each pool using a set of 2749 custom-designed probes (MYcroarray, Inc.) targeting 1510 UCE loci in Hymenoptera (see (Faircloth et al., 2015)). We followed library enrichment procedures for the MYcroarray MYBaits kit (Blumenstiel et al., 2010), except we used a 0.1X concentration of the standard MYBaits concentration, and added 0.7 ľL of 500 ľM custom blocking oligos designed against our custom sequence tags. We ran the hybridization reaction for 24 h at 65 ĄC, subsequently bound all pools to streptavidin beads (MyOne C1; Life Technologies), and washed bound libraries according to a standard target enrichment protocol (Blumenstiel et al., 2010). We used the with-bead approach for PCR recovery of enriched libraries as described in Faircloth et al (2015). We combined 15 ľL of streptavidin bead-bound, enriched library with 25 ľL HiFi HotStart Taq (Kapa Biosystems), 5 ľL of Illumina TruSeq primer mix (5 ľM each) and 5 ľL of ddH2O. We ran post-enrichment PCR using the following thermal profile: 98 ĄC for 45 s; 18 cycles of 98 ĄC for 15 s, 60 ĄC for 30 s, 72 ĄC for 60 s; and a final extension of 72 ĄC for 5 m. We purified resulting reactions using 1.0X speedbeads, and we rehydrated the enriched pools in 22 ľL EB. We quantified 2 ľL of each enriched pool using a Qubit fluorometer (broad range kit). Enrichment was verified by amplifying seven UCE loci (for primers see (Faircloth et al., 2015)) targeted by the probe set. We set up a relative qPCR by amplifying two replicates of 1 ng of enriched DNA from each library at all seven loci and comparing those results to two replicates of 1 ng unenriched DNA for each library at all seven loci. We performed qPCR using a SYBR¨ FAST qPCR kit (Kapa Biosystems) on a ViiATM 7 (Life Technologies). Following data collection, we computed the average of the replicate crossing point (Cp) values for each library at each amplicon, and we computed fold-enrichment values, assuming an efficiency of 1.78 and using the formula 1.78abs(enriched Cp _ unenriched Cp). We then created serial dilutions of each pool (1:200,000, 1:800,000, 1:1.000,000, 1:10.000,000) and performed qPCR library quantification, assuming an average library fragment length of 600 bp. Based on the size-adjusted concentrations estimated by qPCR, we pooled libraries at equimolar concentrations and size-selected for 250Đ800 with a BluePippin (SageScience) where necessary.The pooled libraries were sequenced usingtwo lanes of a 125-bp paired-end Illumina HiSeq run (University of UtahŐs Huntsman Cancer Institute).
SUB2628644
Xylocopa_samarensis_232