Despite its importance in male biology, the human Y chromosome remains poorly characterised at the epigenomic level, with current knowledge restricted to limited CpGs examined by methylation microarrays. Beyond Y-STRs and Y-SNPs, recent forensic studies showed that Y-CpGs can serve as biomarkers, i.e. for age estimation, highlighting the unexploited potential of Y-chromosomal (epi)genetic variation. However, comprehensive profiling approaches across the Y chromosome are lacking. Here, we aimed to develop a custom, large-scale sequencing assay to enable targeted (epi)genomic investigation of the Y chromosome.

To achieve this, we implemented a methylation sequencing workflow based on probe hybridisation for target enrichment (Twist Biosciences). In total, 8,222 probes were designed (hg38, 80-mer), resulting in 29,349 probes targeting both strands and (non-)methylation states using 2× tiling. We targeted 2,034 regions covering 218,702 bp, including existing Y-probes of the Illumina EPIC v2 methylation microarray and 884 previously reported identity Y-SNPs. The workflow consists of genomic DNA fragmentation, library preparation, enzymatic conversion of non-methylated cytosines, followed by probe hybridisation and paired-end sequencing on the Illumina NextSeq500 platform. Sequencing data were processed using an in-house pipeline following Twist guidelines for quality control, alignment, methylation calling, and downstream analysis. To develop our assay, we analysed 10-200 ng of DNA from controls (non-human/female/0-100% methylation), three Genome in a Bottle samples, and sperm cells from 20 males (18-45 years old, European Sperm Bank). We also generated matched Illumina EPIC v2 methylation microarray.

Pilot analyses indicated that ~45% of reads mapped to homologous regions shared between the X and Y chromosomes (pseudo-autosomal region) and were therefore excluded from analysis. On-target rates were ~50% across most samples, whereas the female ‘negative’ control showed the expected predominantly off-target signal (~95%), confirming assay specificity. Most targets achieved high coverage (>100×), with ~32% exceeding 1000×, while 10-20% showed no coverage and ~30% exhibited low to moderate coverage (1–100×). Coverage dropout was primarily observed in AT-rich regions. Results were similar across replicates. To optimise the assay, our efforts focused on refining hybridisation conditions and increasing sequencing output. Finally, Y chromosome-wide methylation levels in sperm were found to be low (10-15%) across DNA inputs and successfully validated against microarray data. Ongoing work includes panel redesign to further improve on-target performance and assay sensitivity.

In conclusion, the developed Y chromosome capture assay demonstrates strong potential for targeted (epi)genomic profiling, laying the foundation for further optimisation and new avenues for male-specific research in forensics and beyond.

Authors

• Thessa Kroes (Department of Clinical Genetics, Maastricht University Medical Centre, Netherlands)
• Femke Ummels (Department of Clinical Genetics, Maastricht University Medical Centre, Netherlands)
• Maartje van de Vorst (Department of Clinical Genetics, Maastricht University Medical Centre, Netherlands)
• Bart de Koning (Department of Clinical Genetics, Maastricht University Medical Centre, Netherlands)
• Arthur van den Wijngaard (Department of Clinical Genetics, Maastricht University Medical Centre, Netherlands)
• Athina Vidaki (Department of Clinical Genetics, Maastricht University Medical Centre; Department of Genetics & Cell Biology, GROW and CARIM Institutes, Maastricht, The Netherlands, Netherlands)