A cost-effective DNA extraction protocol for long-read sequencing in non-model plants

Third-generation sequencing has revolutionized genomics, enabling in-depth analysis of genome sequence, structure, and epigenetic features. Yet, extracting high-quality DNA for long-read sequencing remains a bottleneck-particularly in non-model plants, such as mature trees growing in natural environments, which often contain abundant endogenous compounds that hinder extraction and downstream applications.

We developed an optimized, robust, and cost-effective DNA extraction protocol that yields high-quality DNA suitable for Oxford Nanopore Technologies and PacBio sequencing. Validation across diverse taxa-including six Nothofagus species, gymnosperms endemic to Andean-Patagonian forests, exotic conifers of commercial value, and model plants-demonstrated consistently high DNA purity (A₂₆₀/A₂₈₀ > 1.8, A₂₆₀/A₂₃₀ > 2.0) and fragment sizes ≥30 kbp. Downstream sequencing confirmed suitability for applications requiring long, intact molecules and base modification detection.

Compared to commercial kits and standard protocols, this approach achieved superior DNA integrity and yield without specialized equipment, offering an accessible solution for researchers working with challenging plant species.