Stutter Modeling in Probabilistic Genotyping for Forensic DNA Analysis: A Casework-Driven Assessment

<b>Background:</b> Probabilistic genotyping software has become an essential tool in forensic genetics, particularly for interpreting complex DNA mixtures. Previous studies measured the impact of considering widely divergent statistical approaches in quantifying evidence, both inter- and intra-software. At a much smaller scale, this data-driven study shows how different models implemented on distinct versions of the same tool may affect the results. Among the available tools, EuroForMix stands out as a quantitative, open-source software that models various aspects of the DNA profile, including artefacts like stutter peaks. Its freeware nature allowed the use of both versions 1.9.3. and 3.4.0, between which several updates were made, including the possibility to model both back and forward stutter, compared to only modeling back stutters inputted by the expert in the earlier version. <b>Methods:</b> A total of 156 real casework sample pairs (comprising mixtures with two or three estimated contributors and associated reference) from the Portuguese Scientific Police Laboratory were analyzed using both software versions. The same input data, containing alleles and artefactual peaks, were used to reflect operational conditions. Statistical measurements were compared and further investigated. <b>Results:</b> Most Likelihood Ratio values differed in less than one order of magnitude across versions. However, exceptions were found in more complex samples, such as those with more contributors, unbalanced contributions, or greater degradation. <b>Conclusions:</b> This work emphasizes the relevance of model selection in forensic evidence quantification, even when considering different versions of the same tool. The impact of different models in statistical evaluation depends on several factors, such as sample technical conditions, genotypic profiles, and population distribution.