Methods matter: On the impact of non-transferential variables on reported data of studies on nucleic acid transfer, prevalence, persistence and recovery (TPPR)

“TransAct” is a multi-laboratory collaborative project aimed at establishing foundational work for the rationalization of research on forensic nucleic acid transfer, persistence, prevalence, and recovery (TPPR). A consortium of eight forensic laboratories systematically investigated inter- and intra-laboratory variability in the recovery and quantification of DNA from standardized mixtures of skin cells, buccal cells, and cell-free DNA (as standardized mock touch DNA samples) to improve the understanding of the factors influencing research data on DNA-TPPR. Substantial variability in absolute DNA yields (across two orders of magnitude for identical substrates and four orders of magnitude for different substrates) was observed across participating laboratories, primarily attributable to methodological heterogeneity among labs. Quantification using standard qPCR kits revealed significant differences in measured DNA concentrations depending solely on the method applied, with further variation linked to sampling techniques, and substrates. Mixture proportions of degraded low-template two-person mock touch DNA mixtures (male and female contributors) were assessed via both the ratio of the Y-chromosomal to autosomal qPCR targets and probabilistic genotyping (EuroForMix), revealing discrepancies between the two methods and laboratories. These findings underscore that TPPR data are profoundly influenced by laboratory-specific methodological differences, emphasizing the critical need for experimental standardization and shared resources to enhance the reliability of nucleic acid TPPR research and thereby improving activity level evaluation in forensic genetics. As a step towards harmonizing data collection, this work provides exemplary templates for a detailed documentation in supplementary material, covering both the collection of information on laboratory methods and the standardized display of results to facilitate consistent data reporting and future data pooling.