The Y-chromosome is a powerful piece of DNA in forensic investigations, enabling male-specific profiling in DNA mixtures and supporting familial searching to identify relatives of an unknown perpetrator. Capillary electrophoresis-based Y-typing remains the gold standard in forensic genetics but does not capture sequence-level variation to improve male lineage differentiation.

The introduction of massive parallel sequencing enabled simultaneous analysis of familial Y-chromosomal short tandem repeats (Y-STRs) and evolutionary mutating single nucleotide polymorphisms (Y-SNPs), offering increased resolution for forensic applications. Building on the previously developed CSYseq v1 panel, CSYseq v2 is an enhanced targeted resequencing panel with increased Y-STR and Y-SNP coverage, enabling higher resolution analysis of male lineages in forensic casework. CSYseq v2 sequences 255 Y-STRs, including all Y-STRs from commonly used commercial forensic Y-kits and 50 rapidly mutating (RM) Y-STRs. In addition, the panel targets over 30,000 phylogenetically informative Y-SNPs, alongside more than 14,000 with currently unknown phylogenetic positions. Libraries were prepared using a custom amplicon-based approach and sequenced on Illumina MiSeq and NextSeq 2000 platforms.

Data analysis was performed using optimized bioinformatic pipelines, including FDSTools for Y-STR allele calling and Yleaf for Y-SNP-based haplogroup assignment. The expanded set of 255 Y-STRs aims to improve the power to differentiate closely related males. This is being evaluated through 383  genealogically related males, including father-son pairs, brothers, cousins and more distant relatives (e.g., separated by 4–35 generations), with relationships confirmed both genealogically and genetically. Results are currently being consolidated and will be presented at the conference. Preliminary findings indicate an increased number of Y-STR differences compared to all other Y-sequencing panels, highlighting the added value of the expanded Y-STR set in resolving paternal relatives that remain indistinguishable with conventional Y-STR kits.

In addition, the substantial increase in Y-SNP coverage enables high-resolution haplogroup classification and, for the first time within this framework, the identification of genealogical point mutations (GPMs). These variants arise within defined family pedigrees and allow differentiation between paternal lineages at a genealogical timescale. The detection and validation of such mutations demonstrate the potential of Y-SNPs to complement Y-STR analysis in distinguishing related males from different family branches. 

Overall, CSYseq v2 represents a significant advancement in Y-chromosomal analysis by combining an expanded RM Y-STR panel with unprecedented Y-SNP resolution. This dual approach enhances the discriminatory power for forensic applications and strengthens the ability to differentiate closely related males, directly addressing one of the key challenges in forensic genetics. 

Authors

• Sofie Claerhout (KU Leuven, Belgium) 
• Lara Soeteweye (KU Leuven, Belgium) 
• Floor Reyntien (KU Leuven, Belgium) 
• Haike Vandenabeele (KU Leuven, Belgium) 
• Heleen Coreelman (KU Leuven Kulak, Belgium) 
• Bas Van Haperen (ICMP, International Commission of Missing Persons, Netherlands) 
• Nina Knockaert (KU Leuven, Belgium) 
• Simon Vanpaemel (KU Leuven, Belgium) 
• Kris Van der Gaag (Netherlands Forensic Institute (NFI), Netherlands) 
• Pedro de Carvalho Braga Ilídio Silva (KU Leuven Kulak, Belgium) 
• Tom Van Maerken (AZ Groeninge, Belgium) 
• Celine Vens (KU Leuven Kulak, Belgium) 
• Ellen Decaesteker (KU Leuven Kulak, Belgium) 
• Titia Sijen (Netherlands Forensic Institute (NFI), Netherlands) 
• Ronny Decorte (KU Leuven, UZ Leuven, Belgium)