Xerces blue
Sequencing libraries were prepared following a protocol for genomic shotgun library preparations of Hawaii Drosophila specimens (Tin et al. 2014). This protocol is specifically designed to treat degraded DNA of ancient museum specimens. Based on a Bioanalyzer measurement, the ancient DNA was already sufficiently fragmented; therefore, we omitted the shearing step. In addition to the published protocol, 200 ng of the DNA isolate was dephosphorylated for 1 h at 37 C with FAST Alkaline phosphatase (Thermo Scientific), then deactivated for 10 min at 75 C. The dephosphorylated DNA was then denatured for 3 min at 95 C before being immediately put on ice. The single-stranded DNA was treated with a terminal transferase (TdT) and GTP (Takara) according to the manufactures protocol. The reaction was incubated for 30 min at 37 C before being heat inactivated for 10 min at 70 C. The complimentary strand of the single-stranded DNA was synthesized using the Klenow Fragment (NEB) and CCCCC-oligos for 3 h at room temperature before being inactivated for 20 min at 75 C. The DNA was further treated with T4 polymerase (NEB) for 15 min at 12 C to remove or fill-in 3 and 5 overhangs, respectively, before purified on a MiniElute column (Qiagen). Adaptors were designed as outlined in Tin et al. (2014) and ligated to the DNA fragments before being PCR amplified and cleaned by Dynabeads MyOne Carboxylic Acid beads (Invitrogen). Our produced sequencing library was quality and quantity controlled on a Bioanalyzer (Agilent Technologies) and with qPCR (KAPA Biosystems) according to the user manual. The sequencing library was loaded onto the Illumina MiSeq platform and 150 bp single-end sequenced.