cytochrome oxidase subunit I amplicon sequencing of fruit flies: adult whole body
For amplicon sequencing, a 6-bp barcode sequence was attached to the 5 end of both primers. Each individual was set up with 3 PCR replicates and the PCR reaction mixture was 25l, including 3ng total DNA, 12.5l 2 Q5 High-Fidelity Master Mix (New England Biolabs, Ipswich, MA, U.S.A.), 1.25L (10M) of both forward and reverse primers and the rest supplemented with PCR water. The thermocycling profiles were 98 C for 30 s; 35 cycles of 98 C for 30 s, 51 C for 30 s, and 72 C for 30 s; and 72 C for 2 min. 3.5l of PCR products were used for electrophoresis on 1% agarose gel, and the three positive PCR products of the same specimen were mixed and purified using MagicPure Size Selection DNA Beads II (TransGen, Beijing, China). A DNA library was constructed mixing PCR products of 10 specimens and performed VAHTS Universal Pro DNA Library Prep Kit of pair-end Illumina Miseq (2  303) sequencing (Genewiz, Suzhou, China).