Analysis of uni and bi-parental markers in mixture samples: Lessons from the 22nd GHEP-ISFG Intercomparison Exercise

Area of Science:

• Forensic Science
• Genetics
• Molecular Biology

Background:

• The Spanish and Portuguese-Speaking Working Group of the ISFG (GHEP-ISFG) has conducted annual proficiency tests for forensic DNA laboratories since 1992.
• These exercises, coordinated by the INTCF, include kinship and forensic modules at Basic and Advanced Levels.

Purpose of the Study:

• To evaluate the performance of forensic DNA laboratories in interpreting complex DNA samples, specifically mixtures.
• To identify discrepancies and challenges in the analysis of autosomal, sex-chromosomal STRs, and mitochondrial DNA (mtDNA).

Main Methods:

• Analysis of autosomal and sex-chromosomal short tandem repeats (STRs) and mitochondrial DNA (mtDNA) from two mixture samples (saliva/blood and saliva/semen).
• Evaluation of results from the 2014 GHEP-ISFG Intercomparison Exercise (IE).
• Assessment of allele calls, drop-out/drop-in events, micro-variant detection, contributor proportions, and mtDNA heteroplasmy reporting.

Main Results:

• Discrepancies were observed in autosomal STRs (4.7–6.5%), X-STRs (7.8–15.4%), and Y-STRs (0–1.2%).
• Drop-out/drop-in alleles and varying criteria for minor peak inclusion were primary error sources.
• Significant disagreements occurred in mtDNA analysis, particularly for mixture M4, with issues in heteroplasmy reporting and unexpected profiles.

Conclusions:

• Despite extensive experience, GHEP-ISFG exercises reveal persistent challenges in forensic DNA mixture interpretation.
• Results underscore the necessity for enhanced research and targeted training to improve mixture analysis accuracy among forensic practitioners.