Unrecognised DNA Degradation in Flash-Frozen Genetic Samples in Natural History Collections.

Optimal preservation of tissues from the field to long-term cryo-storage is paramount to securing genetic resources for research needs. DNA preservation techniques vary, with flash freezing currently considered the gold standard in tissue preservation. However, flash freezing tissue samples in the field presents challenges, necessitating a more comprehensive understanding of the quantity and quality of preserved DNA from different techniques in archival collections. We compared metrics from DNA extractions from field-collected amphibian, squamate and bird tissues from archival collections that were flash-frozen in liquid nitrogen or fixed in either ethanol or tissue lysis buffer prior to archival cryopreservation. We also included DNA extracted from tissues of known liquid nitrogen tank failures to provide a baseline of DNA degradation under the very worst-case scenario. Flash-frozen tissues often preserved higher yields of DNA, but peak fragment size, the percentage of fragments larger than 10 kb and DNA integrity numbers were all significantly reduced compared to tissues first preserved in fixative buffers. This pattern was observed across independent samples and between flash-frozen and buffer-preserved pair replicates. Degradation seen in flash-frozen tissues was also distinct to tissues from known tank failures. We suggest that degradation in flash-frozen tissues occurred during shipping, sample sorting/accession or during subsequent subsampling when tissues may partially or fully thaw, exposing DNA to damaging freeze-thaw processes. By contrast, tissues in fixative buffers were likely protected from freeze-thaw damage. This study highlights that using multiple field preservation methods and minimising freeze-thaw cycles for flash-frozen tissues may provide the most robust protection against the DNA degradation sources encountered by field collections.