Can DNA withstand the test of time? Exploring degradation across storage conditions

The integrity of DNA extracts is a critical factor in forensic genetics, particularly when DNA represents the sole source of evidentiary value in criminal investigations. Although technologies such as mini-STRs have improved the amplification of low-quality samples, DNA degradation remains a significant limitation, often resulting from environmental exposure and handling conditions. Heat, light, humidity, microbial contamination, and inadequate storage infrastructure may compromise DNA quality and limit its suitability for downstream analyses. While best practice guidelines recommend low-temperature storage, operational constraints, equipment failures, and inconsistent protocols frequently result in DNA extracts being stored under suboptimal conditions, including room temperature. The present study systematically investigates the long-term preservation of DNA extracts stored under non-ideal and fluctuating temperature conditions, including simulated interruptions to the cold chain. DNA extracts from buccal swabs from two individuals, and their mixture in a 1:15 ratio, were monitored over extended storage periods at -20 °C, + 4 °C, and + 22 °C, as well as under uncontrolled temperature conditions, to assess changes in DNA concentration, degradation indices, and performance in downstream analyses. Selected samples also underwent next-generation sequencing (NGS) after 600 days of storage at different temperatures and conditions to evaluate data quality, including ancestry and phenotype inference. The objectives are to: (a) define optimal preservation conditions for different sample types; (b) evaluate the impact of DNA degradation on STR and NGS results; (c) identify the limitations of NGS in the analysis of degraded DNA; (d) develop evidence-based recommendations for the storage and management of forensic DNA extracts. Results showed that DNA extracts are stable over extended periods also in suboptimal storage conditions, supporting the feasibility of long-term preservation and subsequent re-analysis of extracts in cold-cases, or of samples obtained in emergency non-ideal situations. Evaporation of extracts stored at room temperature affects DNA concentration, but does not compromise DNA integrity or downstream genetic analyses. Interruptions in the cold chain did not result in an immediate collapse of DNA integrity; however, prolonged exposure to the new storage temperature was found to gradually influence DNA preservation. For NGS analyses, DNA quantity, rather than degradation per se, represents the primary limiting factor for successful SNP profiling. Finally, phenotypic and biogeographic ancestry predictions are robust across storage conditions. The expected outcomes include enhancing forensic laboratory practices, improving the reliability of genetic evidence in both current and cold cases, and contributing to the development of updated procedural and legal guidelines within the field.