Development and validation of a 154-plex MPS STR panel for Illumina and Nanopore: a comprehensive tool for forensic genetic analysis

Capillary electrophoresis (CE)-based STR typing, while long established as the gold standard, is constrained by limited allele resolution and multiplexing capacity—unable to resolve variation within length-identical alleles or to simultaneously capture autosomal, X-, and Y-chromosomal STRs in a single reaction. This restricts its utility in complex kinship analysis, sex-related cases, and trace or irreplaceable samples.In this study, we developed a 154-plex STR panel comprising 66 autosomal, 57 Y-chromosomal, and 31 X-chromosomal loci, implemented on Illumina sequencing platform. Following SWGDAM guidelines, the system was rigorously validated using forensic standard DNA (9947A, 9948, and 2800M), 110 unrelated individuals representing blood and other body fluids, and non-human controls. Performance assessments addressed repeatability, sensitivity, inhibitor tolerance, species specificity, concordance with CE typing, and population genetic parameters.The panel demonstrated robust and reproducible performance: complete and accurate STR profiles were obtained at ≥ 0.5 ng DNA input, with >94% accuracy down to 12.5 pg. In DNA mixtures, minor contributor alleles at 10% proportion were reliably recovered. Detection rates exceeded 90% under mild-to-moderate degradation (DI ≤ 6.34), with amplicons <260 bp exhibiting superior resilience. High tolerance to common PCR inhibitors (e.g., 200 ng/µL humic acid, 100 ng/µL tannic acid, and 100 µM haemoglobin) was observed, with bilirubin showing no inhibitory effect. No cross-species amplification was detected, confirming high human specificity. Concordance with CE-derived profiles at overlapping loci was substantial, and sequence-based polymorphisms—including flanking SNPs—enhanced resolution among length-identical alleles. Population genetic analysis of 110 individuals confirmed high power of discrimination across all marker sets. Preliminary evaluation on Nanopore platform further demonstrated concordant performance and potential for rapid, field-deployable applications.Overall, this MPS-based 154-plex STR panel constitutes a robust, high-resolution, and database-compatible system that integrates complementary autosomal and sex-chromosomal markers into a single assay. It substantially enhances forensic analytical capacity across diverse and complex applications, extending beyond conventional laboratory settings.