Nearly two decades on paper: DNA quantity and quality in buccal samples stored on FTA cards

Flinders Technology Associates (FTA™) cards are widely used for the collection and long-term storage of reference DNA samples in forensic databases; however, systematic evaluation of DNA quantity, integrity, and downstream genotyping performance after extended storage remains limited. In this study, buccal cell samples stored on FTA cards for up to 17 years for nuclear short tandem repeat (STR) analysis and up to 20 years for mitochondrial sequencing were evaluated using both extracted and direct-punch workflows. DNA quality and STR performance were assessed using the Investigator® 24plex GO! and Yfiler™ Plus systems, stratified by storage-time category and amplicon-size class. DNA degradation increased with storage duration, whereas PCR inhibition remained minimal. Autosomal STR signals peaked at intermediate storage times and declined after ≥8 years, falling below levels observed in newly collected samples. In contrast, Y-STRs demonstrated a later and more gradual increase in signal intensity, with a modest decline at extended storage intervals that remained above initial levels. Signal reduction in autosomal STRs was more pronounced in intermediate and long amplicons, whereas Y-STR loci showed similar behavior across fragment sizes. Mitochondrial genome sequencing performance was unaffected by storage duration, supporting the long-term stability of mitochondrial DNA on FTA cards. These results indicate that buccal DNA stored on FTA cards remains suitable for nuclear STR analysis for at least 17 years, with mitochondrial genome sequencing extending successfully to 20 years, while storage-associated effects appear assay- and workflow-dependent rather than uniformly driven by DNA quantity or fragment length.