Development and validation of an MPS-based 513-Plex SNP identity panel for degraded forensic samples

Degraded samples pose a challenge in routine forensic practice. The commonly used short tandem repeat markers are not optimally suitable for the analysis of degraded samples because of their structural complexity and locus length. By contrast, single nucleotide polymorphisms (SNPs), characterised by their single base mutation feature, enable the design of short amplification fragments, conferring an advantage in detecting mutations in degraded samples. Hence, our team has developed a multiplex amplification system for individual identification of degraded samples, encompassing 507 autosomal SNP loci, five Y-InDel loci, and one amelogenin sex determination locus. The amplification fragment lengths in this multiplex system range from 81 to 116 bp. The forensic applicability of this panel was validated through sequencing analysis of 201 samples. Among these were 30 degraded samples (simulated degraded samples: heat degradation and ultrasonic fragmentation; formalin-fixed, paraffin-embedded samples). The results indicated that the genotyping accuracy of all loci included in this panel remained at 100% for samples with 0.1 ng of DNA input and severe degradation. Sensitivity experiments revealed that at a DNA input of only 31.25 pg, the locus detection rate reached 100%, with genotype accuracy of 92.5%. Based on population data analysis, the total discrimination power of this system reached 1-5.513 × 10^-143. Furthermore, we included forensic case samples encompassing semen, saliva, menstrual blood, 10-year-old bloodstain cards, and 12-year-old bloodstain cards in the validation studies. The results demonstrated 100% genotyping accuracy across all sample types. Additional, validation data confirmed the system's species specificity (Homo sapiens-specific) and tolerance to inhibitors including humic acid, heme, ethylene diamine tetraacetic acid and indigo (up to 200 µM). In conclusion, this system can serve as a novel tool for the analysis of degraded samples in forensic work.