AHE of  Nigromyia sp.1
The DNA was fragmented by sonication with a Bioruptor Pico (Diagenode, USA), using 50 L of the DNA extractions in 0.1 ml tubes (Cat. No. C30010015), targeting a modal fragment size of 350 base pairs. Libraries were built using the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645L) with NEBNext Multiplex Oligos for Illumina (96 Index Primers) (NEB #E6609L), with two-sided size selection around a mode of 480 base pairs. Target enrichment through hybridization followed the Agilent SureSelectXT Target Enrichment Protocol (Version C2), with a modification at the end whereby the clean, target-enriched DNA suspension was incubated at 95 C for 5 minutes, then immediately put onto the magnetic separator to take off the enriched supernatant. The probe set used the published AHE Diptera Probe set and targeted 559 loci. Enriched libraries were amplified using Phusion Hot Start II High-Fidelity DNA Polymerase (Thermo Fisher Scientific, F549L). PCR cycling consisted of an initial denaturing step at 98 C for 30 s, followed by 8 cycles of denaturing at 98 C for 10 s, annealing at 60 C for 30 s, elongation at 72 C for 30 s, and a final elongation step at 72 C for 10 min.