Sperm cells play a central role in forensic genetics, particularly in sexual assault casework, where their detection and interpretation are critical for identifying perpetrators in multi-donor mixtures. Beyond conventional DNA profiling, sperm cells harbour multiple layers of biological variation, including sequence, structural, and DNA methylation variation, which remain largely unexplored in forensic contexts despite their potential to enhance human and cell-type identification. Comprehensive genome-wide characterization of these features has been limited by short-read sequencing technologies. In contrast, long-read sequencing (LRS) by Oxford Nanopore Technologies (ONT) enables simultaneous detection of genetic and epigenetic variation while improving access to difficult genomic regions, including repetitive and GC-rich sequences. Here, we generated the first-of-its-kind, high-resolution, (epi)genomic map of human sperm cells using ONT sequencing to uncover novel variation relevant to human and cell identity.

Sperm cells were collected from 20 male individuals (between 18-45 years old) via the European Sperm Bank. High-molecular-weight genomic DNA (2–3 µg) was isolated, fragmented (~20 kb), and sequenced on the ONT PromethION 24 platform. Data were processed using ECHO, an in-house pipeline for integrated (epi)genome and repeatome analysis. Genome-wide profiling included sequence, structural, and DNA methylation variation across chromosomes, genomic features, and repetitive DNA classes. Variation profiles were validated using publicly available datasets, compared across cell types, and assessed using matched Illumina EPIC v2 methylation microarray data.
LRS-generated high-quality data (average read quality Q=16.04) with consistent genome-wide coverage across autosomes enabled comprehensive detection of genetic and epigenetic variation. Methylation profiles were validated using known imprinted and sperm-specific differentially methylated regions (DMRs) and showed strong concordance with Illumina data. Comparative analyses identified novel inter-individual variation, including sperm-specific DMRs, methylation quantitative trait loci (mQTLs), structural variants, and repeat-associated variation. Notably, we identified previously uncharacterized CpG-containing short tandem repeats (STRs) with bimodal methylation patterns and allelic variation, demonstrating the value of LRS for resolving complex (epi)genomic features. Ongoing work focuses on validation using targeted sequencing approaches.

In conclusion, this study demonstrates how LRS enables integrated analysis of genetic and epigenetic variation, including within previously inaccessible repetitive regions. By uncovering novel biomarker classes such as methylation-informative STRs, our findings highlight the potential of LRS (epi)genomics to increase discriminative power and allow simultaneous inference of both human identity and tissue origin. Altogether, this approach represents a promising step toward next-generation forensic profiling of complex biological evidence.

Authors

• Mariza Panoutsopoulou (Department of Clinical Genetics, Maastricht University Medical Center; Department of Genetics & Cell Biology, GROW Institute, Maastricht University, Netherlands)
• Leena Putzeys (Department of Clinical Genetics, Maastricht University Medical Center; Department of Genetics & Cell Biology, GROW Institute, Maastricht University, Netherlands)
• Thessa Kroes (Department of Clinical Genetics, Maastricht University Medical Center, Netherlands)
• Jenny van Dongen (Department of Biological Psychology, Amsterdam Public Health Institute, Vrije Universiteit Amsterdam, Netherlands)
• Daniel van den Hove (Department of Psychiatry and Neuropsychology, Mental Health and Neuroscience Research Institute (MHeNs), Netherlands)
• Athina Vidaki (Department of Clinical Genetics, Maastricht University Medical Center; Department of Genetics & Cell Biology, GROW Institute, Maastricht University, Netherlands)