Introducing the Y chromosome ancestral reference sequence - Improving the capture of human evolutionary information

Abstract

20 Reference sequences are essential for reproducible genetic analyses but are often chosen 21 without regard to evolutionary relevance within the analyzed species. The human Y 22 chromosome (chrY) is widely used in evolutionary studies, yet current references represent 23 evolutionarily young sequences, which can lead to misleading variant calling. To address this 24 issue, we constructed a Y-chromosomal ancestral-like reference sequence (Y-ARS) to 25 improve the detection of evolutionarily informative variants on the Y chromosome. 1 bioRxiv preprint doi: https://doi.org/10.1101/2025.05.07.652589; this version posted May 7, 2025. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 26 The Y-ARS was constructed by applying a weighted maximum parsimony approach to human 27 and primate Y chromosome sequences. To benchmark the performance of the Y-ARS, 40 28 chrY short-read sequences from diverse haplogroups were aligned to Y-ARS and existing 29 references (GRCh37, GRCh38, and T2T-CHM13). Overall, the Y-ARS yielded the highest and 30 most consistent number of SNPs per sample (mean=1197; SD=105), while other references 31 yielded on average fewer variants (mean=866–968) and showed greater variability across 32 samples (SD=457–531) depending on their phylogenetic distance from the reference. 33 Additionally, alignments to the Y-ARS resulted in calling solely SNPs with evolutionarily 34 derived alleles, while alignments to other references resulted in calling on average 44% SNPs 35 with ancestral alleles. 36 This study demonstrates how the existing reference sequences fail to capture the full range of 37 evolutionary information on the chrY. The Y-ARS improves capturing evolutionary information 38 on the chrY, making it a valuable resource for various evolutionary applications, such as 39 TMRCA estimations and phylogenetic analyses. Finally, alongside the Y-ARS, we provide a 40 publicly available tool, polaryzer, to annotate variants as ancestral or derived in pre-aligned 41 chrY data. 42 43 Key words: Y-ARS, ancestral state reconstruction, chrY, Y-chromosomal haplogroups, 44 derived state, polaryzer 45 Significance statement 46 Using current reference sequences results in calling genetic variants without information on 47 whether the variant is ancestral or arose later in the species’ history, which is of interest for 48 evolutionary studies. Here, we tackle this problem for the human Y chromosome by 49 introducing the Y-chromosomal ancestral-like reference sequence, Y-ARS. The Y-ARS 50 overcomes this issue by calling only evolutionarily derived variants on the Y chromosome in a 2 bioRxiv preprint doi: https://doi.org/10.1101/2025.05.07.652589; this version posted May 7, 2025. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 51 reproducible way, which can directly be used for various downstream analyses in evolutionary 52 genetics. 53