Combination of Sample Preservation Approaches and DNA Extraction Methods for Long-Read Sequencing of Nudibranchs' Genomes.

With the increasing interest in whole genome sequencing of eukaryotes, it is becoming evident that selecting the most suitable high molecular weight DNA extraction method is crucial for maximizing the benefits of long-read technologies. However, the DNA of many species cannot be processed immediately at the sampling site due to the remoteness of the location, necessitating tissue preservation that may affect DNA fragment size. This study aimed to identify the most suitable combination of four tissue preservation approaches and six DNA extraction methods to ensure high molecular weight DNA. A single Peltodoris atromaculata (Nudibranchia) specimen was sliced into ∼30 mg sub-samples, ensuring consistency across 24 preservation-extraction combinations processed in triplicates. Samples were either stored at 4°C, dried at room temperature, flash-frozen in liquid nitrogen, or preserved in ethanol and stored at -20°C. Afterward, they were processed using five commercially available kits specific for high molecular weight DNA extraction, as well as a custom DNA extraction protocol. Three aspects of DNA quality were evaluated: total yield, fragment size distribution, and availability of DNA for amplification. Most preservation-extraction combinations yielded optimal results in only some of the three DNA quality aspects. We identified six combinations suitable for long-read sequencing: a custom CTAB-based extraction protocol applied to frozen samples, Wizard (Promega) and Nanobind (PacBio) kits for both frozen and ethanol-preserved samples, and the ethanol preservation paired with Monarch (NEB) kits. The suitability of the six selected combinations was confirmed by PacBio sequencing, producing a total yield of 3.6 Gbp (3.2x estimated genome coverage). The results indicate that the success of high molecular weight DNA extractions is influenced by preservation methods. Although tested on nudibranchs, these findings are highly useful for genomic studies of other organisms, which may need to be preserved in remote locations before being transported to the laboratory for processing.