UCE target enrichment of Messor spp
Sequencing libraries were prepared according to the protocol outlined by Faircloth et al. (2012, 2015) for Ultra-Conserved Elements capture, with modifications detailed in Cruaud et al. (2019). DNA was fragmented to a size of 400 bp using the Bioruptor Pico (Diagenode). Subsequent steps included end repair, 3-end adenylation, adapter ligation, and PCR enrichment, all performed using the NEBNext Ultra II DNA Library prep kit for Illumina (NEB). Barcoded adapter pairs were utilized, containing amplification and Illumina sequencing primer sites, along with a 5 or 6 bp nucleotide barcode for sample identification. Pools of 16 samples were created at an equimolar ratio. UCE capture was accomplished by enriching each pool using the Insect Hymenoptera 2.5K version 2, Ant-Specific probe set (Branstetter et al., 2017) and MYbaits kits (MYcroarray, Inc.). The manufacturers protocol (MYbaits, user manual version 3) was followed. The hybridization reaction was conducted for 24 hours at 65C. Post-enrichment amplification was performed on beads using the KAPA Hifi HotStart ReadyMix. Enriched libraries were quantified with Qubit, an Agilent Bioanalyzer, and qPCR using the Library Quantification Kit Illumina/Universal from KAPA (KK4824). The libraries were then pooled at an equimolar ratio. Paired-end sequencing (2x300bp) was carried out on an Illumina MiSeq platform at UMR AGAP (Montpellier, France).