UCE target enrichment of Bombus_mckayi_BLX1677 [raw reads]
We extracted DNA from the mid and hind legs of specimens using a Zymo Quick-DNA Miniprep Plus extraction kit and stored extracts in -80C freezers at PIRU. We sheared the DNA fragments to target fragment sizes of 400 to 600 base pairs using a Q800R2 acoustic sonicator (Qsonica, Newtown, CT, U.S.A.). We varied shearing times from 0 seconds to 120 seconds with a 10 seconds on, 10 seconds off pulsing pattern. Samples with small fragment sizes were sheared for less time and samples with large fragment sizes were sheared for more time. Once sonicated, we purified the DNA samples using a homemade paramagnetic bead solution (Rohland and Reich 2012). We prepared Illumina sequencing libraries using Kapa Hyper prep kits and custom 8 bp dual indexing adapters (Glenn et al. 2019). We amplified the libraries using 12 cycles of PCR, cleaned the amplified DNA using 1.0 to 1.2x SPRI beads to remove contaminants and fragments smaller than 200 bp, and quantified the DNA using Qubit. Samples with low measured volumes of DNA were re-amplified for 14 to 16 PCR cycles from an aliquot of the pre-PCR library. We enriched the samples using an existing UCE bee-ant specific baitset (bee-ant-specific Hym-v2, Branstetter et al. 2017, Grab et al. 2019) identified and optimized for use in the order Hymenoptera. The baitset was developed using seven genomes from hymenopteran species, including two species from the bee families Apidae (the family that contains all bumble bees)  and Halictidae. We enriched the pooled libraries following a combination of the Arbor Biosciences v3.02 protocol (enrichment day 1) and a protocol based on Blumenstiel et al. (2010, available at ultraconserved.org). We pooled up to ten samples per library at equimolar concentrations for enrichment. Finally, we repeated the PCR amplification, purification, and quantification steps previously described for the pooled enriched samples. Enriched pools were combined into a final sequencing pool and sent to Novogene Inc. for sequencing on an Illumina HiSeq X instrument (PE150).