Developmental validation of a novel pentameric STR panel for enhanced kinship and mixture resolution.

Distant kinship testing remains a major challenge in forensic genetics, as the discrimination power provided by routine STR kits is often insufficient to resolve complex relationship scenarios. To address this limitation, we developed a novel pentameric STR panel, composed of five newly implemented highly polymorphic loci together with Penta E, intended to complement conventional markers in both kinship and criminal casework. A total of 304 DNA samples from Iberian individuals were genotyped to characterize the panel. The five novel markers showed high levels of polymorphism, with 18-30 alleles detected per locus, and exhibited discrimination power comparable to that of highly informative STRs such the SE33 and D12S391. The performance of the panel in kinship analysis was assessed through both simulations and real casework involving different genetic degrees of relatedness. When combined with established STR kits, the novel multiplex improved the resolution of kinship cases involving relatives up to second degree. Its applicability to criminal casework was also evaluated. Compared with conventional tetrameric STR systems, the pentameric loci showed lower stutter ratios, which may facilitate mixture interpretation. In addition, an allele-specific stutter threshold was established to improve discrimination between stutter peaks and true minor-contributor alleles in mixed DNA profiles. The high heterozygosity of the novel loci also contributed to a more accurate inference of the number of contributors in DNA mixtures. Finally, developmental and forensic validation studies, together with an optimized protocol for profiling degraded and low-template DNA samples, confirmed the suitability of the panel for routine forensic applications.