Comparative Evaluation of OSIRIS and GenoProof Suite Against GeneMapper—Software for STR Profile Interpretation

Background/Objectives: Accurate short tandem repeat (STR) profile interpretation is crucial for reliable forensic DNA analysis, but artifacts and degraded DNA frequently complicate evaluations. While GeneMapper® is the industry standard, high licensing costs restrict multi-user accessibility. This study comparatively evaluates the open-source software OSIRIS v2.16 and the commercial platform GenoProof Suite v1.0.3 against GeneMapper v5 to assess their allele-call concordance, artefact handling, and practical functionality as viable and cost effective alternatives. Methods: We used a dataset of 113 human reference DNA profiles (3400 alleles), including 68 concordant profiles and 45 flagged cases with artifacts, mixtures, or degraded DNA. Raw .fsa capillary electrophoresis data were generated on a SeqStudio™ Genetic Analyzer (Applied Biosystems, Bethesda, MA, USA) using the NGM Select™ kit (Applied Biosystems, Bethesda, MA, USA). We analysed the samples independently across all three platforms with directly comparable analytical parameters standardised wherever possibleand compared them side by side for allele designation, peak heights, and artifact detection. Results: Both alternative platforms achieved over 99% allele-calling concordance with the independently reviewed reference dataset. OSIRIS achieved an allele call concordance rate of 99.32%, automatically designating off-ladder alleles and microvariants without manual intervention. GenoProof Suite reached an allele call concordance rate of 99.12% but flagged off-ladder alleles, requiring manual review. OSIRIS consistently reported higher, non-uniform relative fluorescence unit (RFU) values due to its adaptive baseline modeling and double-Gaussian curve-fitting. However, OSIRIS supported manual editing of peaks recognised during automated processing but did not allow the manual addition of signals absent from its Locus Edit table, whereas GenoProof Suite and GeneMapper® provided this functionality, which was particularly relevant for low-level mixture profiles. Conclusions: OSIRIS and GenoProof Suite represent highly viable alternatives to GeneMapper®. OSIRIS provides an automated, free, and open-source option highly suited for laboratories with budget constraints or for educational training, whereas GenoProof Suite offers flexible modular features and robust manual editing capabilities. Neither software was validated as a fully autonomous expert system.