Internal validation of the Mini25A system: The six-dyes labelled kit for forensic degraded samples

Short tandem repeats (STRs) represent the gold-standard genetic markers for individual identification and kinship analysis in forensic genetics. However, the relatively large amplicon sizes of STR loci in conventional commercial kits often hinder their utility for analyzing aged or degraded biological samples, where fragmented DNA is prevalent. MiniSTR technology addresses this limitation by redesigning primers to target regions closer to the STR core repeats, thereby shortening amplicon lengths and enabling robust genotyping of compromised samples. Despite this advantage, most currently available miniSTR kits incorporate a limited number of loci, which translates to insufficient cumulative forensic efficiency to meet the rigorous demands of forensic casework. In this study, we conducted a comprehensive performance validation of the commercial NHID Mini25A kit, evaluating key forensic metrics including species specificity, genotyping precision, platform concordance, allelic peak height balance, and so on. The validation results demonstrated that the NHID Mini25A kit exhibits stringent species specificity, yielding unambiguous genotyping profiles exclusively from human DNA. Allele calling precision was high, with standard deviations consistently below 0.1, and the kit showed full compatibility with both ABI 3130 and 3500 Genetic Analyzer platforms. Moreover, the kit maintained favorable heterozygote balance, as well as robust intra-dye and inter-dye peak height balances. It achieved a high sensitivity of 0.031 ng, with no evidence of allele dropout even at this low DNA input. Additionally, the NHID Mini25A kit displayed a notable tolerance to common forensic inhibitors and demonstrated strong efficacy in analyzing mixtures across a range of contributor ratios, successfully identifying alleles derived from minor contributors. Most notably, compared with the conventional 25A casework kit, the NHID Mini25A kit exhibited substantially superior performance for different degree of degraded samples, retaining the ability to effectively genotype ≥ 88% of its targeted loci. In conclusion, the NHID Mini25A kit demonstrated stable and reliable performance across all critical validation parameters, rendering it an efficient and practical tool for the detection and analysis of challenging degraded samples in forensic genetics research.