Whole mitochondrial genome seq of Himantura leoparda
Genomic DNA was fragmented to ~500 bp using a Bioruptor Plus sonicator (Diagenode) at 4C (low power, 5 cycles: 3 cycles, cooling, 2 cycles; 30/90 s ON/OFF). Fragmentation was verified by 1% agarose gel electrophoresis. Size selection (>250 bp) was performed using 1.2 Sera-Mag Carboxylate-Modified Magnetic Beads (Cytiva) via left-side selection. All subsequent steps utilized SPRI-based on-bead processing. Fragments were blunt-end repaired (25C for 15 min, 12C for 5 min), ligated to P5/P7 adapters (Integrated DNA Technologies; 22C, 30 min), and subjected to fill-in reaction (37C, 20 min). Libraries were eluted in low TE buffer. Amplification was performed using KAPA HiFi Taq Ready Mix (2) with 1218 cycles (98C/45 s; 1218 [98C/15 s, 65C/30 s, 72C/45 s]; 72C/1 min), adjusted to achieve 610 ng/L. Indexing PCR was performed similarly. Final libraries (0.10.9 ng/L) were quantified using a Qubit 2.0 Fluorometer, pooled at 10 nM, and sequenced on an Illumina MiniSeq platform using the High Output Kit (300 cycles) with 2150 bp paired-end reads.