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

Area of Science:

• Forensic Science
• Genetics
• Molecular Biology

Background:

• Degraded DNA samples present significant challenges in forensic identification due to limitations of traditional short tandem repeat (STR) markers.
• Single nucleotide polymorphisms (SNPs) offer advantages for degraded samples due to their small amplicon sizes, facilitating mutation detection.
• A need exists for robust molecular tools capable of analyzing compromised forensic evidence.

Purpose of the Study:

• To develop and validate a multiplex amplification system for individual identification using single nucleotide polymorphisms (SNPs) and Y-InDel loci.
• To assess the system's performance on degraded and challenging forensic samples.
• To evaluate the sensitivity, specificity, and discriminatory power of the novel SNP panel.

Main Methods:

• Development of a multiplex system comprising 507 autosomal SNP loci, 5 Y-InDel loci, and 1 amelogenin locus.
• Validation using sequencing analysis on 201 samples, including 30 simulated and real-world degraded samples (heat, ultrasonic, FFPE).
• Testing sensitivity down to 31.25 pg DNA input and assessing tolerance to common forensic inhibitors.

Main Results:

• 100% genotyping accuracy was achieved for all loci with 0.1 ng DNA input and severe degradation.
• High locus detection (100%) and genotype accuracy (92.5%) were observed at 31.25 pg DNA input.
• The system demonstrated a high total discrimination power (1-5.513 × 10^-143), 100% accuracy on diverse forensic samples, species specificity, and inhibitor tolerance.

Conclusions:

• The developed multiplex SNP system is highly effective for the individual identification of degraded forensic samples.
• This novel tool offers superior performance compared to traditional markers for compromised evidence.
• The system provides a reliable and sensitive solution for routine forensic casework involving challenging samples.