CSYseq: The first Y-chromosome sequencing tool typing a large number of Y-SNPs and Y-STRs to unravel worldwide human population genetics

Author summary Around 95% of the male-specific Y-chromosome (chrY) is non-recombining and therefore inherited in a conserved manner from father to son. It can therefore serve as a powerful marker for interdisciplinary genetic-genealogical research as it provides a strong link between genetic information and a family tree or pedigree. While Y-chromosomal short tandem repeats (Y-STRs) discriminate close paternal kinships, single nucleotide polymorphisms (Y-SNPs) enables the identification of far evolutionary ancestry. Unfortunately, an extensive chrY-specific sequencing panel combining a large number of familial Y-STRs and evolutionary Y-SNPs was not yet available. Therefore, chrY is rarely included in research projects and not often linked to a genealogical, history-demographical or life science database. In this way, the importance of chrY still remains not yet fully understood. Massive parallel sequencing (MPS) allows the simultaneous analysis at sequence level of Y-SNPs and Y-STRs with variable mutation rates in a large number of males. However, up until today, no commercial kit is exploiting the full potential that MPS offers on chrY. Therefore, we developed the 'CSYseq', which is the first extensive chrY-specific sequencing panel. The CSYseq simultaneously identifies 9,014 slow mutating Y-SNPs to identify evolutionary ancestry, and 202 rapid mutating Y-STRs to investigate paternal relationships. We validated and optimized the panel through the analysis of 130 males distributed over 65 families. This novel MPS panel is useful for biogeographical identity and ancestry analysis, together with Y-chromosome profiling for the identification of patrilineages and discrimination of closely related males. As the CSYseq includes a very diverse set of markers that can be easily interpreted, it is interesting for different interdisciplinary applications within evolutionary, population, molecular, medical and forensic genetics. Male-specific Y-chromosome (chrY) polymorphisms are interesting components of the DNA for population genetics. While single nucleotide polymorphisms (Y-SNPs) indicate distant evolutionary ancestry, short tandem repeats (Y-STRs) are able to identify close familial kinships. Detailed chrY analysis provides thus both biogeographical background information as paternal lineage identification. The rapid advancement of high-throughput massive parallel sequencing (MPS) technology in the past decade has revolutionized genetic research. Using MPS, single-base information of both Y-SNPs as Y-STRs can be analyzed in a single assay typing multiple samples at once. In this study, we present the first extensive chrY-specific targeted resequencing panel, the 'CSYseq', which simultaneously identifies slow mutating Y-SNPs as evolution markers and rapid mutating Y-STRs as patrilineage markers. The panel was validated by paired-end sequencing of 130 males, distributed over 65 deep-rooted pedigrees covering 1,279 generations. The CSYseq successfully targets 15,611 Y-SNPs including 9,014 phylogenetic informative Y-SNPs to identify 1,443 human evolutionary Y-subhaplogroup lineages worldwide. In addition, the CSYseq properly targets 202 Y-STRs, including 81 slow, 68 moderate, 27 fast and 26 rapid mutating Y-STRs to individualize close paternal relatives. The targeted chrY markers cover a high average number of reads (Y-SNP = 717, Y-STR = 150), easy interpretation, powerful discrimination capacity and chrY specificity. The CSYseq is interesting for research on different time scales: to identify evolutionary ancestry, to find distant family and to discriminate closely related males. Therefore, this panel serves as a unique tool valuable for a wide range of genetic-genealogical applications in interdisciplinary research within evolutionary, population, molecular, medical and forensic genetics.