The prevalence of highly repetitive sequences within the human Y chromosome has prevented its complete assembly to date1 and led to its systematic omission from genomic analyses. Here we present de novo assemblies of 43 Y chromosomes spanning 182,900 years of human evolution and report considerable diversity in size and structure. Half of the male-specific euchromatic region is subject to large inversions with a greater than twofold higher recurrence rate compared with all other chromosomes2. Ampliconic sequences associated with these inversions show differing mutation rates that are sequence context dependent, and some ampliconic genes exhibit evidence for concerted evolution with the acquisition and purging of lineage-specific pseudogenes. The largest heterochromatic region in the human genome, Yq12, is composed of alternating repeat arrays that show extensive variation in the number, size and distribution, but retain a 1:1 copy-number ratio. Finally, our data suggest that the boundary between the recombining pseudoautosomal region 1 and the non-recombining portions of the X and Y chromosomes lies 500 kb away from the currently established1 boundary. The availability of fully sequence-resolved Y chromosomes from multiple individuals provides a unique opportunity for identifying new associations of traits with specific Y-chromosomal variants and garnering insights into the evolution and function of complex regions of the human genome.
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All data generated and used in this study were derived from lymphoblast lines available from the Coriell Institute for Medical Research for research purposes (https://www.coriell.org/). Further details are provided in Supplementary Table 1. All data generated by the HGSVC (PacBio HiFi, ONT-UL, Hi-C, RNA-seq, Iso-seq and Bionano Genomics optical genome maps) are available at the International Nucleotide Sequence Database Collaboration (INSDC) under the following NCBI project IDs: PRJEB58376, PRJNA988114, PRJEB41077, PRJEB39684 and PRJEB39750. The HPRC (https://humanpangenome.org/) year 1 PacBio HiFi, ONT long-read sequencing and Bionano Genomics optical mapping data files are available at INSDC (PRJNA701308). Further details are provided in Supplementary Table 1. Existing testis Iso-seq data from seven individuals are available from the European Nucleotide Archive (ENA) under accessions SRX9033926 and SRX9033927. The GEUVADIS expression data are available through ArrayExpress under accession E-GEUV-3. The Genome in a Bottle (GIAB) data can be downloaded from ENA (PRJNA200694). Large supplementary data files such as the assembled genomes are available online (ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/data_collections/HGSVC3/working/20230412_sigY_assembly_data).
Project code implemented to produce the assemblies and the basic quality control/evaluation statistics is available at GitHub (https://github.com/marschall-lab/project-male-assembly). All scripts written and used in the study of the Yq12 subregion are available at GitHub (https://github.com/Markloftus/Yq12).
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Funding was provided by National Institutes of Health (NIH) grants U24HG007497 (to C. Lee, E.E.E., J.O.K. and T.M.), U01HG010973 (to T.M., E.E.E. and J.O.K.), R01HG002385 and R01HG010169 (to E.E.E.), and GM123312 (to S.J.H. and R.J.O.); the German Federal Ministry for Research and Education (BMBF 031L0184 to J.O.K. and T.M.); the German Research Foundation (DFG 391137747 to T.M.); the German Human Genome-Phenome Archive (DFG (NFDI 1/1) to J.O.K.); the European Research Council (ERC Consolidator grant 773026 to J.O.K.); the EMBL (to J.O.K. and P. Hasenfeld); the EMBL International PhD Programme (to W.H.); the Jackson Laboratory Postdoctoral Scholar Award (to K.K.); NIH National Institute of General Medical Sciences (NIGMS R35GM133600 to C.R.B.; 1P20GM139769 to M.K.K. and M.L.) and the National Cancer Institute (NCI) (P30CA034196 to C.R.B. and P.A.A.); U24HG007497 (P. Hallast, F.Y., Q.Z., F.T. and J.Y.K.); NIGMS K99GM147352 (to G.A.L.); and Wellcome grant 098051 (to C.T.-S.). This work was also supported, in part, by the P30 CA034196 grant from the NCI. E.E.E. is an investigator of the Howard Hughes Medical Institute. We thank A. Rhie and A. Phillippy for coordination and discussions; Y. Xue for discussions and advice throughout the project; J. Wood and the members of the Genome Reference Informatics Team at the Wellcome Sanger Institute for suggestions and feedback on assembly evaluation; L. Skov for advice and sharing his scripts for gene conversion detection; the members of the HPRC (https://humanpangenome.org) for making their data publicly available; the staff at Clemson University for their allotment of compute time on the Palmetto Cluster; staff at the Center for Information and Media Technology at Heinrich Heine University Düsseldorf and the Scientific Services at the Jackson Laboratory, including the Genome Technologies Service for their assistance with the work described herein and Research IT for providing computational infrastructure and support and the members of the Phillippy laboratory (NIH/NHGRI) for their Verkko support; and the people who contributed samples as part of the 1000 Genomes Project.
E.E.E. is a scientific advisory board member of Variant Bio, Inc. C. Lee is a scientific advisory board member of Nabsys and Genome Insight. The following authors have previously disclosed a patent application (no. EP19169090) relevant to Strand-seq: J.O.K., T.M. and D.P. The other authors declare no competing interests.
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Extended data figures and tables
a. Overview of the Y chromosome. A three-way comparison of sequence identity between GRCh38 Y, NA19317 (E1b1a1a1a1c1a1a3a1-CTS8030) and the T2T Y (excluding Yq12 and PAR2 subregions), highlighting substantial differences in the size and orientation of some subregions. b. Focus on Yq12. Sequence identity heatmaps of the Yq12 subregion for six contiguously assembled samples (HG01890, HG02666, HG01106, HG02011, HG00358 and HG01952), two samples (NA19705 and HG01928) with a single gap in the Yq12 subregion (gap location marked with asterisk) and the T2T Y using 5kb window size. c. Focus on TSPY repeat array. Sequence identity heatmaps of ~20.3-kbp TSPY repeat units for three males highlighting putative expansion events harbouring both single and multiple repeat units. Red shades from lighter to darker indicate sequence identity from 99–100%, respectively, while white fill indicates sequence identity <99%. The last copy on the right is the single separate repeat unit containing the TSPY2 gene. See Fig. S22 for heatmaps of all samples. d. Dotplots of the TSPY repeat array for HG02666 with 5 kbp of flanking regions showing identical matches of 2, 5, and 10 kbp in size indicating regions with high sequence identity. See Fig. S25 for additional examples.
Extended Data Fig. 2 Distribution of genetic variants across the Y chromosome and repeat elements in PAR1, XDR1 and XTR1 subregions.
a. Distribution of variant sizes for SVs (≥ 50 bp, top), Indels (< 50 bp, middle), and SNVs (bottom) with the Y chromosome coloured by subregion. High peaks in heterochromatin are apparent for SVs, but are absent in SNVs and indels. b. Repeat element distribution across 10 samples with contiguously assembled PAR1 regions and the T2T Y. Repeat elements on sense (+) and antisense (−) strand are shown, coloured according to repeat class. Extensive differences in size can be seen between samples, especially in the satellite arrays located close to the telomere (in dark red), and substantial differences in repeat element composition in PAR1 vs. the male-specific XDR1 and XTR1 regions. The locations of PAR1, XDR1 and XTR1 subregions in each individual are shown in black, red and black, respectively. Please note that the maroon colour of the “Unknown” elements close to the telomere is caused by significant clustering of those elements. DNA: DNA repeat elements, snRNA: small nuclear RNA, tRNA: transfer RNA, rRNA: ribosomal RNA, srpRNA: signal recognition particle RNA, scRNA: small conditional RNA, RC: rolling circle.
a. Inversions identified in the AZFc/ampliconic 7 subregion. Top - comparison between the T2T Y and select de novo assemblies, bottom - GRCh38 Y and the de novo assemblies (see Fig. S34 for details on AZFc/ampliconic 7 subregion composition). Potential NAHR path is shown below the dotplot. b. Inverted duplication affecting roughly two thirds of the 161 kbp unique ‘spacer’ sequence in the P3 palindrome, spawning a second copy of the TTTY5 gene and elongating the LCRs in this region. A detailed sequence view reveals a high sequence similarity between the duplication and its template, and its placement in Y phylogeny supports emergence of this variant in the common ancestor of haplogroup E1a2 carried by NA19239, HG03248 and HG02572 (Fig. 3a).
a. A schematic distribution of individual RBMY1 gene copies (filled rectangles) within analysed Y chromosome assemblies (42 + T2T + GRCh38). The RBMY1 gene copies are located in four primary regions (NA19239 carries a partial duplication of gene region 2 and the composition of HG02666 suggests at least one inversion within the RBMY regions). Fill colours refer to the assigned network community (NC) and indicates a similar sequence (Methods). Assembly of this region was not contiguous in HG03065 (brown line) and was not included in the analysis. b. A secondary directed network showing connections between NCs with the most similar consensus sequences. An edge pointing from one node to a second node indicates that the second node was the first’s closest match (i.e., most similar sequence; ties are allowed and shown as multiple edges stemming from a node). The width of the edge represents the sequence similarity between two nodes (i.e., NC consensus sequence similarity; thicker means fewer SNVs). The node size is representative of the total edges pointing to the node. c. RBMY1 phylogenetic analysis of exonic nucleotide sequences. Shown is the unrooted phylogenetic tree of RBMY1 genes constructed using a maximum likelihood approach (Methods). This tree is rooted at the midpoint with the total count of RBMY1 copies shown on the right. The scale bar represents the average number of substitutions per site. RBMY1 copies located in regions 1 and 2 (primarily dark blue, orange, dark/light green, and pink) distinguish themselves from those located downstream in regions 3 and 4 (primarily light blue and purple copies).
a. TSPY array visualization of each sample with contiguous assembly in this region. Individual TSPY gene copies are shown (rectangles), and their colour is based on the assigned network community (NC) (Methods). Sample names with black rectangles (NA19331, HG03732 and HG03492) carry the IR3/IR3 inversion and were re-oriented for visualization. Asterisks within individual gene copies indicate possible gene conversion (GC) or recombination (R) events unique to that gene copy. If a GC/R event is shared by an NC an asterisk is shown in the NC legend rectangle. The TSPY2 gene copy is shown as a red rectangle. b. A secondary directed network showing the sequence similarity between NC consensus sequences. An edge pointing from one node to a second node indicates that the second node was the first’s closest match (i.e., most similar sequence; ties are allowed and shown as multiple edges stemming from a node). The width of the edge represents the sequence similarity between two nodes (i.e., NC consensus sequence similarity; thicker means fewer SNVs). The node size is representative of the total edges pointing to the node. c. TSPY phylogenetic analysis of exonic nucleotide sequences. Shown is the unrooted phylogenetic tree of TSPY genes constructed using a maximum likelihood approach (Methods). This tree is rooted at the midpoint and the total count of TSPY copies is shown on the right. The scale bar represents the average number of substitutions per site. The early split/rise of NC1 within the tree, in conjunction with the secondary directed network and manual comparison of TSPY sequences (as well as their presence across all lineages) suggests that NC1 TSPY copies represent the ancestral TSPY gene sequence.
Extended Data Fig. 6 DNA methylation patterns as determined from the ONT data across the three contiguously assembled Y chromosomes.
Methylation patterns for samples: a. HG1890, b. HG02666 and c. HG00358. The three dot plots (in grey) show the smoothed DNAme levels, in 5 kbp windows for visualization, in beta-scale ranging from 0 (not methylated) to 1 (methylated). The locations of Yq12 repeat arrays (DYZ18, 2.7kb-repeat, 3.1kb-repeat, DYZ1 and DYZ2) and the Y-chromosomal subregions are shown below as bar plots.
Extended Data Fig. 7 Functional analyses on the Y chromosome with DNA-methylation, RNA expression and HiC information as anchored to GRCh38 Y.
a. The top three panels show DNA-methylation levels and variation over the studied chromosomes (n = 41). In black (top dot plot) the average methylation is shown, in green (middle dot plot) the variation in DNAme levels across the studied genomes. The bottom boxplot represents the DNA methylation segmentation using PycoMeth-seg (Methods). In grey shades 2,861 methylation segments, and in red shades the 340 significantly differentially methylated segments (DMS). The CpG sites that fall in a DMS are coloured in a lighter shade in the top two dot plots, the dot plots are in beta-scale, ranging from 0 (not methylated) to 1 (methylated). b. Average insulation scores (top) and variance of insulation scores between any two samples (bottom) across 40 samples with Hi-C data with 10 kbp resolution. Regions with lower insulation scores are more insulated and more likely to be topologically associating domain (TAD) boundaries, while regions with higher scores are more likely to stay inside TADs (the regions between the two adjacent TAD boundaries). The y-axis represents the average insulation scores ranging from −2 (most insulated) to 2 (least insulated) and the variance insulation scores ranging from 0 (no variance) to 8 (more variance). c. The Geuvadis-based gene-expression analysis, shown are the 205 genes on the Y chromosome (grey shades), the 64 genes expressed in the Geuvadis LCLs (blue shades), of which 22 are differentially expressed (red shades, Supplementary Results ‘Functional analysis’ for additional details).
a. Organization of the chromosome Y centromeric region from 21 genomes representing all major superpopulations. The structure (top), α-satellite HOR organization (middle), and sequence identity heat map (bottom) for each centromere is shown and reveals the presence of novel HORs in over half of the centromeres. Note - the sizes of the DYZ3 α-satellite array are shown on top as determined using RepeatMasker (Methods). b. Genetic landscape of the Y-chromosomal pericentromeric region for three select samples (see Figs. S47–S48 for all samples). The top panel shows locations and composition of the pericentromeric region with repeat array sizes shown for each Y chromosome (the DYZ3 α-satellite array size as determined using RepeatMasker, Methods). The middle panel shows (UL-)ONT read depth and bottom sequence identity head maps generated using the StainedGlass pipeline74 (using a 5 kbp window size).
An overview of the Bray-Curtis distance/dissimilarity of k-mer abundance profiles for individual DYZ18 (grey), 3.1-kbp (red), 2.7-kbp (blue) and DYZ1 (black) repeats versus their consensus sequence. The Yq11/transition region/Yq12 are shown for each of the seven samples with a completely assembled Yq12 subregion. Lighter colours indicate less distance/dissimilarity (i.e., more similar) k-mer abundance profiles compared to their consensus sequence. Results indicate that arrays located on the proximal and distal boundaries of the Yq12 subregion contain repeats with k-mer abundance compositions less similar to their consensus sequence (i.e., more diverged). The size of individual lines is a function of the length of the repeat. The repeat unit orientation (above = sense, below = antisense) was determined based on RepeatMasker annotations of satellite sequences within repeats (Methods).
An overview of the divergence of individual DYZ2 subunits for a. samples with completely assembled Yq12 subregion (HG01890, HG02666, HG01106, HG02011, T2T Y, HG00358, HG01952), and b. the two most closely related genomes (NA19317 and NA19347) with incompletely assembled Yq12 subregions. The size of individual lines is a function of the length of the repeat. The repeat unit orientation (above = sense, below = antisense) was determined based on RepeatMasker annotations of satellite sequences within repeats (Methods). A higher divergence was observed within the subunits located in arrays at the proximal and distal ends of the Yq12 subregion. Additionally, DYZ2 subunits located near the boundaries of individual arrays tend to be more diverged than those located centrally. Between the closely related genomes, the divergence of DYZ2 repeats within the shared DYZ2 arrays are highly similar.
Supplementary Results 1–9 providing more quality control and in-depth analyses of gene content, inversions, the Yq12 region and putatively functional consequences of the Y variation; Supplementary Figs. 1–74, complementing all analyses in the main text and in the Supplementary Results; and Supplementary References 1–46.
Supplementary Tables 1–61, complementing all analyses in the main text and in the Supplementary Results. The file includes a short description of each table in the table of contents (the first table sheet in the document).
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Hallast, P., Ebert, P., Loftus, M. et al. Assembly of 43 human Y chromosomes reveals extensive complexity and variation. Nature 621, 355–364 (2023). https://doi.org/10.1038/s41586-023-06425-6
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