Monozygotic (MZ) twins present a unique challenge in forensics due to their (nearly) identical genomes, making their differentiation based on conventional DNA profiling methods impossible. Recent studies suggest that early postzygotic mutations and environment-driven epigenetic discrepancies accumulated over time could offer new avenues for their individual identification. Yet, these differences are rare or technically difficult to measure in trace material. A major source of variation lies within the human repeatome, encompassing tandem repeats (TRs) and transposable elements (TEs), which is highly polymorphic and subject to somatic (epi)mutations, thus presenting a promising source of forensic twin-discriminating biomarkers. This study aimed to develop a novel genome-wide approach for investigating these so far hidden layers of biological variation in MZ twins using long-read sequencing (LRS). 

We sequenced the whole (epi)genomes from blood (n=3) and saliva (n=1) of four MZ twin pairs (2 female/2 male, 28-61 years old) using Oxford nanopore sequencing. As input we used 2 and 3 µg of blood and saliva DNA, respectively, following the ligation sequencing DNA V14 kit. For data processing we developed and implemented a novel pipeline (ECHO) for joint haplotype-resolved (epi)genomic analysis including a wide range of variants (SNPs, SVs, CpGs) and repeat classes (TRs and TEs). Overall, the obtained sequencing read quality was high (Q-score>20), with sufficient post-processing coverage for both blood (~40×) and saliva (~20×). Blood sequencing data resulted in higher read length distributions (N50: ~10Kb) compared to saliva (N50: 1-2Kb).  

For each MZ twin pair we identified putative twin-differentiating variants including rare SNPs, thousands of methylation differences as well as a few larger SVs and differentially methylated CpG islands. Twin divergence varied per twin pair and tissue, and were heavily dependent on filtering. Notably, confirming our hypothesis, several candidates originated from repetitive elements, i.e. unique TR polymorphisms and TE insertions, particularly those from the Alu family. Putative twin-specific TE insertions varied in size (250-2,000 bp) and followed allele-specific or mosaic patterns. Ongoing work focuses on refining our filtering strategy, and importantly, validating the most promising identified variants using targeted sequencing approaches. 

Our study proposing repeatome profiling via LRS represents a groundbreaking approach in forensics. Our pilot results show promise for future MZ twin differentiation and broaden the scope of forensic investigations by leveraging previously unexplored (epi)genomic variation. Future efforts to expand our cohort including additional tissues will enable further progress towards establishing an innovative human identification strategy in complex casework involving MZ twins. 

Authors

• Athina Vidaki (Department of Clinical Genetics, Maastricht University Medical Centre; Department of Genetics and Cell Biology, GROW Institute, Maastricht University, Maastricht, The Netherlands, Netherlands) 
• Mariza Panoutsopoulou (Department of Clinical Genetics, Maastricht University Medical Centre; Department of Genetics and Cell Biology, GROW Institute, Maastricht University, Netherlands) 
• Leena Putzeys (Department of Clinical Genetics, Maastricht University Medical Centre; Department of Genetics and Cell Biology, GROW Institute, Maastricht University, Netherlands) 
• Thessa Kroes (Department of Clinical Genetics, Maastricht University Medical Centre, Netherlands) 
• Nicole Marancenbaum (Department of Clinical Genetics, Maastricht University Medical Centre, Netherlands) 
• Wim Loneus (Department of Clinical Genetics, Maastricht University Medical Centre, Netherlands) 
• Sofie Claerhout (Forensic Biomedical Sciences, Department of Imaging and Pathology, KU Leuven; Interdisciplinary Research Facility Life Sciences, KU Leuven Kortrijk, Belgium) 
• Aimee Paulussen (Department of Clinical Genetics, Maastricht University Medical Centre, Netherlands)