UCE target enrichment of Timulla_baucis_TIM045: whole organism
DNA extraction was performed using a High Pure PCR Template Preparation Kit (Roche Diagnostics, Indianapolis, Indiana, USA). Entire adult specimens were primarily used for extraction except for rare species in which a single mesoleg and metaleg were removed and partly crushed. The entry point for extraction material into the specimens was typically the resulting pin hole in the mesosoma after removing the pin. Following extraction, specimens were rinsed using 95% ethanol and remounted on pins to allow for future study. Extracted DNA was quantified using a Qubit 3.0 Fluorometer. The extracted DNA was prepared for shearing to a target concentration of 50 ng/100 µL. The samples were then sheared into fragments ranging between 400 and 600 base pairs using a Qsonica Q800R2 (Qsonica L.L.C., Newtown, Connecticut, USA). Library preparation was performed using a KAPA Hyper-Prep Kit (Roche Sequencing and Life Science, Wilmington, Massachusetts, USA) and Illumina TruSeq-style adapters (Glenn et al., 2019). Libraries were pooled using 10 samples at equimolar ratios resulting in pools with 500 ng total DNA to be used for targeted UCE enrichment. Each pool was enriched using a standardized protocol based on myBaits Hybridization Capture for Targeted NGS Manual v.4.01 and Branstetter et al. (2017a). Enrichment was performed using a custom Ant-Specific probe set (myBaits UCE Hymenoptera 2.5Kv2A) which targets 2524 conserved loci and 16 nuclear exons (Branstetter et al., 2017b) (available from Arbor Biosciences (Ann Arbor, Michigan, USA)). The probes were hybridized to the libraries at 65°C for an incubation period of 24 hours. Enrichment success was determined via qPCR using a Bio-Rad CFX96 system. Each pool was quantified using the qPCR results and was further pooled into a single pool containing 110 total libraries. Sequencing was performed using an Illumina HiSeq X Ten system at Novogene Corporation Inc. (Chula Vista, California, USA).