Nondestructive DNA extraction from specimens and bulk samples preserved in DESS solution for DNA barcoding

DNA barcoding of small organisms often requires significant damage or destroy specimens. To address this, the development of nondestructive DNA extraction methods that maintain specimen morphology is crucial. Here, we present a protocol using the supernatant of DESS preservation solution (20% DMSO, 250 mM EDTA, saturated NaCl), which conserve both the morphological characteristics and DNA of biological samples long-term. This method successfully conducted nondestructive barcoding of nematodes preserved in DESS and stored 10 years at room temperature. The protocol also applies to bulk environmental samples, where sediment and seagrass collected in the field are immediately preserved in DESS. This enables the subsequent isolation and individual nondestructive barcoding of meiofauna and diatoms from the preserved environmental samples in the laboratory. This study describes a nondestructive DNA extraction method from nematode specimens and small organism in environmental samples stored in DESS preservation solution. DNA could be extracted from just 500 µl of the DESS supernatant without impacting morphology, and the extracted DNA was amplified using universal primers targeting the barcoding region. Sequencing was performed using Sangar and Nanopore platforms, confirming the obtained sequences were consistent with the taxonomic classifications based on specimen morphology. This study developed a nondestructive DNA extraction protocol from DESS preservation solution, enabling DNA barcoding while maintaining specimen morphology intact.The protocol successfully extracted DNA from 10-year-old nematode specimens preserved in DESS at room temperature, demonstrating its effectiveness for long-term specimen preservation.We validated the method for bulk environmental samples, showing that meiofauna and diatoms isolated from sediment around seagrass roots can be DNA barcoded without damaging the specimens.The nondestructive approach preserved valuable voucher specimens for future morphological studies while still allowing molecular analysis, addressing a critical need in taxonomic research.Comparison of Sanger and Nanopore sequencing technologies revealed their complementary strength, with Sanger being more accurate for homopolymer regions and Nanopore better for identifying mixed species in environmental samples. This study developed a nondestructive DNA extraction protocol from DESS preservation solution, enabling DNA barcoding while maintaining specimen morphology intact. The protocol successfully extracted DNA from 10-year-old nematode specimens preserved in DESS at room temperature, demonstrating its effectiveness for long-term specimen preservation. We validated the method for bulk environmental samples, showing that meiofauna and diatoms isolated from sediment around seagrass roots can be DNA barcoded without damaging the specimens. The nondestructive approach preserved valuable voucher specimens for future morphological studies while still allowing molecular analysis, addressing a critical need in taxonomic research. Comparison of Sanger and Nanopore sequencing technologies revealed their complementary strength, with Sanger being more accurate for homopolymer regions and Nanopore better for identifying mixed species in environmental samples.