Development and validation of a STR and SNP multiplex detection system (88 STRs and 348 SNPs) using massively...

Area of Science:

• Forensic genetics
• Molecular biology
• Human identification

Background:

• Short tandem repeats (STRs) are standard in forensic DNA analysis.
• Single nucleotide polymorphisms (SNPs) offer complementary forensic information.
• Massively parallel sequencing (MPS) enhances DNA marker detection resolution.

Purpose of the Study:

• To develop and validate a comprehensive MPS-based multiplex system for simultaneous STR and SNP analysis.
• To assess the forensic applicability of the developed system according to SWGDAM guidelines.
• To evaluate the system's performance in sensitivity, accuracy, inhibitor robustness, degraded DNA, and mixture analysis.

Main Methods:

• Development of a multiplex MPS assay for 88 STRs and 348 SNPs.
• Validation following SWGDAM guidelines, including sensitivity, accuracy, inhibitor, degraded DNA, and mixture studies.
• Population genetics analyses for cumulative power of discrimination (CPD) and cumulative probability of exclusion (CPE).

Main Results:

• High sensitivity achieved: 100% STR profiles from ≥0.125 ng, 100% SNP profiles from ≥0.0625 ng.
• Excellent accuracy (99.78% concordance with CE) and robustness against PCR inhibitors.
• Successful genotyping from degraded DNA (<500 bp) and reliable mixture analysis (1:1, 1:3 ratios), including Y-STR detection in 99:1 mixtures.
• High population genetics metrics: CPD and CPE > 0.999999.

Conclusions:

• The validated MPS multiplex system offers high sensitivity, accuracy, and reliability for forensic human identification.
• This system represents a significant advancement in forensic DNA analysis methodology.
• The comprehensive panel of STRs and SNPs provides extensive applicability and practical value in forensic genetics.