Development and validation of a 55-Plex X-STR NGS panel for forensic casework: Population genetics, mutation...

X-chromosome short tandem repeats (X-STRs) are valuable genetic markers in forensic genetics, particularly for complex kinship analysis and the analysis of male mixtures, due to their unique inheritance pattern. However, the application of X-STRs has been limited by the relatively small number of loci included in commonly used capillary electrophoresis (CE)-based assays, and the lack of sequence-level polymorphism information provided by CE. This study addresses these limitations by developing and validating a novel next-generation sequencing (NGS) panel comprising 55 X-STR markers. The panel was constructed using multiplexed PCR capture and sequenced on Illumina platforms. We performed comprehensive developmental validation studies according to the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, assessing repeatability, concordance, sensitivity, resistance to PCR inhibitors, performance with degraded DNA, analysis of DNA mixtures, and species specificity. The panel demonstrated excellent performance across all validation parameters. A minimum input of 0.5 ng of DNA is recommended for reliable genotyping. The system showed robust resistance to humic acid (up to 7 ng/μL) and hematin (up to 30 μM). Accurate genotyping was maintained even with degraded DNA samples. For DNA mixtures, the minor contributor alleles could be reliably detected up to a 1:19 ratio (male-male) and 1:9 (female-male). Furthermore, we conducted a population genetics survey of 268 individuals (154 females and 114 males) from the Han Chinese population in Hebei Province, including a subset of 128 unrelated individuals. A total of 648 alleles were identified across the 55 loci. No significant deviations from Hardy-Weinberg equilibrium (HWE) or linkage equilibrium (LE) were observed after Bonferroni correction, except for three LD pairs. Population genetic parameters, including power of discrimination (DP) and power of exclusion (PE), were calculated. Furthermore, paternal and maternal mutation rates were estimated based on 51 father-mother-children pedigrees including 102 offspring. Finally, we evaluated the panel^'s ability to distinguish second-degree kinship relationships using both real-world samples (67 pairs) and simulated data (44,000,000 pairs). The results demonstrated the panel^'s enhanced discriminatory power compared to existing X-STR systems, achieving overall accuracies of 80.47 % to 93.20 % in simulated second-degree kinship tests. This 55-plex X-STR NGS panel provides a powerful new tool for forensic casework, particularly for complex kinship analysis, and contributes valuable population data for the Han Chinese population. The study also highlights the importance of considering sequence variation in X-STR analysis and provides insights into X-STR mutation rates.