The principal limitation of forensic Y-STR analysis, which identifies a male lineage rather than an individual man, is being addressed by the discovery and application of rapidly mutating Y-STRs (RM Y-STRs). Due to their higher mutation rates compared to standard Y-STRs used in forensics, RM Y-STRs significantly enhance the ability to differentiate between male relatives. However, some male relatives - particularly closely related ones - remain indistinguishable. Given the design and execution of the two previous RM Y-STR searches that discovered the 26 currently known RM Y-STRs, it is unlikely that future searches will largely increase the number of RM Y-STRs. To address the ongoing forensic challenge of differentiating between male relatives using Y chromosome analysis, this study explorers an alternative approach: Y-chromosomal singe nucleotide variants (Y-SNVs) obtained via whole genome sequencing (WGS). To assess the feasibility of the WGS technology in differentiating closely and distantly related males, we sequenced DNA samples of 24 male individuals belonging to three deep-rooted pedigrees, covering 12 father-son pairs and 72 pairs of distant male relatives separated by 8-15 meioses. Among the 76 meioses analyzed in total, 90 male relative-differentiating Y-SNVs were identified across the approximately 25 Mbp Y chromosome sequence generated per sample. A total of 141 male relative-differentiating Y chromosome mutations were observed when also considering Y-STRs from Yfiler Plus, RMplex, and WGS analyses. Of the 12 father-son pairs, six (50 %) were differentiated by one or more Y-SNVs, and 9 (75 %) with WGS and CE methods combined. All of the 72 pairs of distant male relatives were distinguished both through Y-SNVs and RM Y-STRs. Overall, when compared to RMplex, WGS yielded a 1.7-fold increase in the number of observed mutations in father-son pairs and a 4-fold increase in distantly related males. Our proof-of-principle study demonstrates (i) the feasibility and high value of Y-SNV markers and WGS technology in differentiating both close and distant male relatives
(ii) the superior performance of Y-SNVs from WGS relative to the previously used RM Y-STR markers and RMplex method
and (iii) the enhanced male relative differentiation achieved by combining both marker types and methods. We envision WGS as the method of choice for maximizing male relative differentiation based on Y chromosome information in high-profile criminal cases with male suspects where no autosomal STR profiles are available and where standard Y-STR and RM Y-STR analyses fail to distinguish the suspect from his male paternal relatives.