MuCTAB: An Optimized Protocol for High-Molecular-Weight DNA Extraction from Mucilage-Rich Selenicereus Tissues for Long-Read Sequencing

Extracting high-molecular-weight (HMW) DNA from cactus tissues remains technically challenging due to the abundance of mucilage, pectins, polyphenols, and other metabolites that compromise DNA purity, increase viscosity, and reduce integrity, thereby limiting its suitability for long-read sequencing. This constraint is particularly relevant in Selenicereus megalanthus, a crop of increasing agronomic and genomic importance for which optimized protocols for third-generation sequencing remain limited. Here, we compared four CTAB-based DNA extraction protocols using dehydrated cladode tissue and evaluated DNA quality using NanoDrop spectrophotometry, Qubit fluorometry, agarose gel electrophoresis, and functional validation via sequencing on the Oxford Nanopore PromethION 2 Solo platform. Among the tested methods, our proposed optimized mucilage-adapted CTAB (MuCTAB) protocol, comprising 4% CTAB, 4% PVP-40, 0.5% β-mercaptoethanol, and proteinase K, showed the best overall performance. MuCTAB yielded the highest dsDNA concentration (239.63 ± 34.37 ng/µL), optimal purity ratios (A260/A280 = 1.96 ± 0.05; A260/A230 = 2.01 ± 0.01), and superior DNA integrity. Nanopore validation confirmed its effectiveness, with MuCTAB producing the highest sequencing yield (84.2 Gbp), read count, mean read quality score (Q18.4), read N50 (40.3 kbp), and maximum read length (1.9 Mbp). Overall, MuCTAB represents a low-cost, reproducible, and efficient method for HMW DNA extraction from mucilage-rich pitahaya tissues and shows promising potential for adaptation to other recalcitrant plant species.