UCE target enrichment of Schusterphotopsis_barghesti_EX1831: whole organism
Library preparation, UCE enrichment, and contig assembly were performed in two separate laboratories (Utah State University (EMUS), Logan, UT, U.S.A. and the National Museum of Natural History (NMNH), Washington, D.C., U.S.A.) and the methodologies used in both labs were mostly similar. Any methods that differ between the labs are noted as having been performed at either EMUS or NMNH. DNA extraction was performed using a High Pure PCR Template Preparation Kit (Roche Diagnostics, Indianapolis, IN, U.S.A.) at EMUS or with a standard phenol-chloroform method at Cornell University and later prepared at NMNH. 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 400–600 base pairs using a Qsonica Q800R2 sonicator at EMUS or a Qsonica Q800R sonicator at NMNH (Qsonica L.L.C., Newtown, CT, U.S.A.). Library preparation was performed using a KAPA HyperPrep Kit (Roche Sequencing and Life Science, Wilmington, MA, U.S.A.) and Illumina TruSeq-style adapters (Glenn et al., 2019). Libraries were pooled at equimolar ratios of 10 with up to 500 ng of DNA 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 at EMUS (myBaits UCE Hymenoptera 2.5Kv2A) which targets 2,524 conserved loci (Branstetter et al., 2017b) or the Principal Hymenoptera probe set at NMNH (myBaits UCE Hymenoptera 2.5Kv2P) which targets 2,590 conserved loci (Branstetter et al., 2017a) (both available from Arbor Biosciences (Ann Arbor, MI, U.S.A.)). The probes were hybridized to the libraries at 65C 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 pools of 110 total libraries. The EMUS pools were sequenced using an Illumina HiSeq X Ten system at Novogene (Chula Vista, California, U.S.A.) and the NMNH pools were sequenced using an Illumina HiSeq 2500 system at the Cornell Institute of Biotechnology (BRC) (Ithaca, New York, U.S.A.).