Mitochondrial genome sequencing and assembly of Kadsura coccinea: leaf
Fresh leaf samples of Kadsura coccinea are collected, and high-quality DNA is extracted using a plant DNA extraction kit (DNeasy Plant Mini Kit, Qiagen) and quantified using a fluorometric method (Qubit dsDNA HS Assay Kit, Thermo Fisher Scientific). The extracted DNA is sheared into desired fragment sizes using enzymatic methods (NEBNext dsDNA Fragmentase, New England Biolabs) or mechanical methods (Covaris), followed by end-repair, A-tailing, and adapter ligation using a library preparation kit (NEBNext Ultra II DNA Library Prep Kit, New England Biolabs) to convert it into a sequencing library. After purification with size-selection methods (AMPure XP beads, Beckman Coulter) and optional library amplification (KAPA HiFi HotStart ReadyMix, Roche), the prepared library is assessed for quality and size distribution (Agilent Bioanalyzer or TapeStation) and accurately quantified using qPCR (KAPA Library Quantification Kit, Roche) or fluorometric methods. The library is then loaded onto the Promethion flow cell, which is primed and loaded with necessary reagents (Promethion Sequencing reagents, Oxford Nanopore Technologies), and sequencing run parameters are set up through the Promethion software. Upon completion, base calling and data processing are performed (Guppy, Oxford Nanopore Technologies), followed by trimming and filtering of raw data (NanoFilt, NanoPlot, Porechop).