Whole genome sequencing of Lysandra bellargus: adult male leg
DNA was extracted from a two adult legs usingQiagen extraction kits (56304 QIAamp DNA Micro Kit (50)). DNA was standardised to 100ul of 15ng/ul DNA per sample. DNA was sheared by sonication using a BioRuptor (o6-9 cycles 30s on/ 90s off (water at ~4C)). This was followed by a size selection step using a 0.85x MagNa bead size selection. The bead mix was produced in-house using SPRI beads from GEHealthcare Sera-Mag SpeedBeads Carboxyl Magnetic Beads, hydrophobic (Fisher:65152105050250). After size selection, WGS paired end (150bp) whole genome libraries were prepared using a NEBnext ULTRA II kit. Briefly, DNA was end-repaired following the manufacturers instructions. Illumina adapters were ligated to the end repaired DNA mixture. The library was cleaned using a 1.6x MagNaBead purification. A unique individual barcode was ligatedto each individual using a 3-cycle PCR (denaturation: 98C, 30s x 1 cycle. Denaturation, anneal and extension:98C for 10s, 65C for 75s x 3cycles. Final extension: 65C for 5 minutes). This ensured ligation of the barcode without PCR enrichment of the sample. Finally, the library was cleaned using a 0.9x MagNa Bead cleanup. As the DNA fragments were degraded, no size selection was conducted.