van der Gaag, Kristiaan Jvan Marion, Vincentvan den Berg, Redmar RWeiler, Natalie ECHoogenboom, JerryKal, ArnoudKayser, Manfredde Knijff, PeterLaros, Jeroen FJSijen, TitiaSlooten, Klaas2025-06-262025-06-262025-05-034036766210.1016/j.fsigen.2025.103292https://rivm.openrepository.com/handle/10029/655439In forensic casework, monozygotic twins have always provided a challenge, as routinely used forensic Short Tandem Repeat (STR) profiles are not able to differentiate between the twin individuals. In this study, we applied a method to discriminate between two monozygotic twin brothers in a sexual assault case that is unique and challenging for several reasons: the use of contact stains as evidence, the stains contain DNA from two persons (victim and one of the brothers), have minimal amounts of DNA, and there are PCR inhibiting factors. Despite these challenging factors, we present a successfully solved case in which whole genome sequencing was applied to identify multiple somatic differences between the two brothers. Validation of the developed methods and the identified differences was performed on material provided by the two siblings, before applying the method on two evidentiary stains. A statistical framework was developed to provide a likelihood calculation for this type of analysis in mixed stains. The results were accepted in court and contributed to the conviction of the case suspect. Here we provide the scientific details in order to encourage the use of this approach in more such cases in the future.enCopyright © 2025 Elsevier B.V. All rights reserved.Forensic DNA analysisLikelihood calculationMPSMonozygotic twinsSomatic mutationsTargeted massively parallel sequencingWGSWhole genome sequencingJournal ArticleForensic Sci Int Genet 2025; 78:103292S1872-4973(25)00072-9