Development and Validation of MPS-Based System for Human Appearance Prediction in Challenging Forensic Samples.

Area of Science:

• Forensic genetics
• Human pigmentation
• Molecular biology

Background:

• Forensic DNA phenotyping (FDP) enables trait prediction from crime scene DNA.
• Existing methods may require larger DNA quantities or lack comprehensive marker coverage.

Purpose of the Study:

• To develop and evaluate a multiplex assay for simultaneous prediction of eye, hair, and skin color using the HIrisPlex-S system.
• To assess the forensic suitability and sensitivity of the assay with various sample types and DNA degradation levels.

Main Methods:

• Development of a 41-plex Massively Parallel Sequencing (MPS) assay for HIrisPlex-S markers.
• Sequencing of diverse forensic samples (blood, skeletal remains, touch DNA, saliva) and controls on the Ion Torrent platform.
• Optimization of PCR cycles and library preparation reagents; evaluation of DNA input quantity and degradation impact.

Main Results:

• Reliable DNA profiles obtained with as little as 0.1-5 ng of DNA, including highly degraded samples.
• Successful prediction of eye and hair color in 92.60% and skin color in 85.15% of individuals.
• DNA quantity, not degradation level, was the critical factor for accurate genotyping and phenotyping.

Conclusions:

• The developed 41-plex MPS assay is robust, highly sensitive, and suitable for human pigmentation prediction in forensic genetics.
• The assay provides reliable results even with challenging forensic samples.
• This tool enhances the capabilities of forensic investigations by predicting key donor characteristics.