Development and inter-laboratory validation of the VISAGE enhanced tool for age estimation from semen using...

Area of Science:

• Forensic science
• Epigenetics
• Molecular biology

Background:

• DNA methylation analysis is a key method for estimating chronological age.
• Forensic science requires reliable age estimation from biological evidence like semen.
• Existing forensic assays for semen age prediction are limited.

Purpose of the Study:

• To develop and validate a novel assay for age estimation from semen using massively parallel sequencing.
• To assess the assay's reproducibility, sensitivity, and performance in mimicking casework conditions.
• To develop a streamlined version of the assay for practical forensic application.

Main Methods:

• Development of a new assay targeting 13 candidate CpG sites in two multiplex PCRs.
• Validation by five international consortium laboratories (VISAGE project).
• Testing with DNA methylation controls, extracts, and semen stains on FTA cards.

Main Results:

• The assay achieved high read depths (>1000 reads) across all 13 markers.
• Robust quantification of DNA methylation levels with low standard deviation (2.8%).
• High concordance (approx. 1% absolute difference) between laboratories and sample types.

Conclusions:

• The developed massively parallel sequencing assay is robust and suitable for forensic age estimation from semen.
• A streamlined 5-marker assay shows comparable performance, enhancing applicability.
• This assay advances forensic capabilities for determining age from semen evidence.