Whole mitochondrial genome seq of Himantura leoparda
Genomic DNA libraries were prepared using the Ion Plus Fragment Library Kit (ThermoFisher Scientific) according to manufacturer's protocol (MAN0009847 K.0). DNA was fragmented using a Covaris S2 ultrasonicator (10% duty cycle, 5% intensity, 200 cycles/burst, 260s), followed by AMPure XP bead purification (1.8 volume). Fragments were end-repaired (20 min, room temperature), purified (1.8 AMPure XP), and ligated to IonCode Barcode Adapters (22C, 15 min). Libraries were amplified (8 PCR cycles) and size-selected (~475 bp) using the Pippin Prep system with 2% dye-free gel cassettes. Fragment distribution was verified on a LabChip GX Touch 24 analyzer. Libraries were quantified using the Ion Library TaqMan Quantitation Kit on a StepOnePlus Real-Time PCR System, diluted to 40 pM, and pooled equimolarly. Template preparation was performed on an Ion Chef using the Ion 510/520/530 Chef Kit. Sequencing was conducted on an Ion GeneStudio S5 Prime System with Ion 530 Chips.