Analyzing degraded DNA and challenging samples using the ForenSeq™ DNA Signature Prep kit.

Area of Science:

• Forensic Science
• Genetics
• Molecular Biology

Background:

• Short tandem repeat (STR) typing is standard for forensic human identification using capillary electrophoresis (CE).
• Massively parallel sequencing (MPS) offers increased discriminatory power and can analyze more genetic markers simultaneously.
• MPS technology is advantageous for analyzing challenging, degraded forensic DNA samples.

Purpose of the Study:

• To evaluate Verogen's ForenSeq™ DNA Signature Prep kit for analyzing degraded DNA samples.
• To compare the performance of MPS with CE for forensic identification using challenging samples.
• To assess the reliability and effectiveness of MPS for forensic casework with degraded DNA.

Main Methods:

• Utilized Verogen's ForenSeq™ DNA Signature Prep kit with over 150 genetic markers (STRs and SNPs).
• Tested the kit on serially degraded DNA samples and 24 mock case-type samples (bones, blood cards, teeth).
• Performed repeated experimental runs and triplicate sequencing of pooled libraries for validation.

Main Results:

• The ForenSeq™ kit successfully analyzed degraded DNA samples, yielding 90% of CODIS loci even with severe degradation (DI: 44).
• MPS outperformed a standard CE kit (PowerPlex® Fusion), which obtained only 35% of CODIS loci on severely degraded DNA.
• Dropped-out loci in MPS were associated with long amplicons and low read numbers; Y-STR anomalies were attributed to sequence structures or library preparation, not sequencing artifacts.
• Positive controls showed locus drop-outs, primarily in SNPs, despite passing quality metrics.

Conclusions:

• Verogen's ForenSeq™ DNA Signature Prep kit is effective for analyzing degraded forensic DNA samples.
• MPS technology provides a significant advantage over CE for forensic identification, especially with challenging samples.
• This study supports the validation and adoption of MPS technologies in forensic laboratories.