Abstract
The human Y chromosome has been notoriously difficult to sequence and assemble because of its complex repeat structure that includes long palindromes, tandem repeats and segmental duplications1,2,3. As a result, more than half of the Y chromosome is missing from the GRCh38 reference sequence and it remains the last human chromosome to be finished4,5. Here, the Telomere-to-Telomere (T2T) consortium presents the complete 62,460,029-base-pair sequence of a human Y chromosome from the HG002 genome (T2T-Y) that corrects multiple errors in GRCh38-Y and adds over 30 million base pairs of sequence to the reference, showing the complete ampliconic structures of gene families TSPY, DAZ and RBMY; 41 additional protein-coding genes, mostly from the TSPY family; and an alternating pattern of human satellite 1 and 3 blocks in the heterochromatic Yq12 region. We have combined T2T-Y with a previous assembly of the CHM13 genome4 and mapped available population variation, clinical variants and functional genomics data to produce a complete and comprehensive reference sequence for all 24 human chromosomes.
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Data availability
The T2T-CHM13v2.0 (T2T-CHM13+Y) assembly, reference analysis set, complete list of resources—including gene annotation, repeat annotation, epigenetic profiles, variant-calling results from 1KGP and SGDP, gnomAD, ClinVar, GWAS and dbSNP datasets—are available for download at https://github.com/marbl/CHM13. The assembly is also available from NCBI and EBI with GenBank accession GCA_009914755.4. Annotation and associated resources are also browsable as ‘hs1’ from the UCSC Genome Browser (http://genome.ucsc.edu/cgi-bin/hgTracks?db=hub_3671779_hs1), the Ensembl Genome Browser (https://projects.ensembl.org/hprc/) (assembly name T2T-CHM13v2.0) and NCBI data-hub (https://www.ncbi.nlm.nih.gov/data-hub/genome/GCF_009914755.1/). Potential assembly issues are listed and can be tracked at https://github.com/marbl/CHM13-issues. 1KGP and SGDP short-read alignments and variant calls are available within AnVIL at https://anvil.terra.bio/#workspaces/anvil-datastorage/AnVIL_T2T_CHRY. Original data from the Gerton lab underlying this manuscript can be accessed from the Stowers Original Data Repository at http://www.stowers.org/research/publications/libpb-2358. Sequencing data used in this study are listed in Supplementary Table 1.
Code availability
Custom codes developed for data analysis and visualization are available at https://github.com/arangrhie/T2T-HG002Y, https://github.com/snurk/sg_sandbox and https://github.com/schatzlab/t2t-chm13-chry and are deposited with Zenodo159. Software and parameters used are stated in the Supplementary Methods with further details.
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Acknowledgements
We thank P. Hallast, M. C. Loftus, M. K. Konkel, P. Ebert, T. Marschall and C. Lee for coordination and discussions, J.C.-I. Lee for sharing the GRCh38-Y coordinates used in Y-Finder and members of the Telomere-to-Telomere consortium and HPRC for constructive feedback. This work utilized the computational resources of the National Institutes of Health (NIH) HPC Biowulf cluster (https://hpc.nih.gov). Computational resources were partially provided by the e-INFRA CZ project (no. 90140), supported by the Ministry of Education, Youth and Sports of the Czech Republic and Computational Biology Core, Institute for Systems Genomics, University of Connecticut. Certain commercial equipment, instruments and materials are identified to specify adequately experimental conditions or reported results. Such identification does not imply recommendation or endorsement by the NIST, nor does it imply that the equipment, instruments or materials identified are necessarily the best available for the purpose. We thank the Intramural Research Program of NHGRI, NIH no. HG200398 (A.R., S.N., S.K., M.R., A.M.M., B.P.W. and A.M.P.); NIH no. GM123312 (S.J.H., P.G.S.G., G.A.H. and R.J.O.); NIH no. GM130691 (P.M., M.H.W. and K.D.M.); HHMI Hanna Gray Fellowship (N.A.); NIH no. CA266339 (J.G. and T.P.); NIH no. GM147352 (G.A.L.); NIH nos. HG002939 and HG010136 (R.M.H. and J.M.S.); NIH no. HG009190 (P.W.H., A. Gershman and W.T.); NIH nos. HG010263, HG006620 and CA253481 and NSF no. DBI-1627442 (M.C.S.); NIH no. GM136684 (K.D.M.); NIH nos. HG011274 and HG010548 (K.H.M.); NIH nos. HG010961 and HG010040 (H.L.); NIH no. HG007234 (M.D.); NIH no. HG011758 (F.J.S.); NIH no. DA047638 (E.G.); NIH no. GM124827 (M.A.W.); NIH no. GM133747 (R.C.M.); NIH no. CA240199 (R.J.O.); NIH nos. HG002385, HG010169 and HG010971 (E.E.E.); Stowers Institute for Medical Research (J.L.G. and T.P.); National Center for Biotechnology Information of the National Library of Medicine, NIH (F.T.-N. and T.D.M.); intramural funding at NIST (J.M.Z.); NIST no. 70NANB20H206 (M.J.); and NIH nos. HG010972 and WT222155/Z/20/Z and the European Molecular Biology Laboratory (J.A., P.F., C.G.G., L.H., T.H., S.E.H., F.J.M. and L.S.). RNA generation was supported by NIST no. 70NANB21H101 and NIH no. 1S10OD028587; the Ministry of Science and Higher Education of the Russian Federation, St. Petersburg State University, no. PURE 73023672 (I.A.A.); the Computation, Bioinformatics, and Statistics Predoctoral Training Program awarded to Penn State by the NIH (A.C.W.); and Achievement Rewards for College Scientists Foundation, The Graduate College at Arizona State University (A.M.T.O.). E.E.E. is an investigator for HHMI.
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V.A.S. is retired from the Institute of Molecular Genetics. Assembly was carried out by S.N., S.K. and M.R. Validation was performed by A.R., S.K., M.A., A.V.B., G.F., A.F., A.M.M., J.M., A.M., L.F.P., D.P., F.J.S., K.S., P.M., J.M.Z. and K.D.M. ChrY haplogroups were determined by A.R. and A.C.W. Alignment was done by C.-S.C., M.D., R. Harris, M.R.V. and K.D.M. Satellite annotation was performed by N.A., I.A.A., G.A.L., F.R., V.A.S. and K.H.M. N.A., J.G. and T.P. carried out FISH. Repeat annotation was done by S.J.H., P.G.S.G., G.A.H., R.M.H., J.M.S. and R.J.O. Retro-elements were dealt with by R. Halabian and W.M. Non-B DNA was dealt with by M.H.W. and K.D.M. Gene annotation was undertaken by A.R., M.D., P.F., C.G.G., L.H., M.H., J.H., T.H., F.J.M., T.D.M., S.L.S., A.S. and F.T.-N. A.R., R. Harris, W.T.H., P.M., M.T. and K.D.M. dealt with ampliconic genes. Structural annotation was performed by A.R., M.C., H.L., P.M. and K.D.M. Epigenetic analysis was performed by A.R., P.W.H., A. Gershman, W.T. and A.M.W. Mappability was performed by A.M.T.O., M.A.W. and J.M.Z. Non-B DNA was dealt with by M.H.W. and K.D.M. Variants and liftover were carried out by A.R., D.J.T., S.K., J.A., N.-C.C., M.D., E.G., A. Guarracino, N.F.H., W.T.H., S.E.H., S.H., R.C.M., N.D.O., M.E.G.S., L.S., M.R.V., S.Z., J.M.Z., E.E.E. and A.M.P. A.R., S.L.S., B.P.W. and A.M.P. dealt with contamination. Data generation was carried out by M.J., R.K.K., A.P.L., J.K.L., C.M., B.M.M., K.M.M., H.E.O., F.J.S. and Y.Z. Data management was undertaken by A.R., M.D., M.J. and J.K.L. Computational resources were sourced by R.J.O., M.C.S. and A.M.P. A.R., S.N., M.C., S.J.H., D.J.T., N.A., I.A.A., N.-C.C., E.G., J.G., P.G.S.G., A. Guarracino, R. Halabian, W.M., J.M., T.P., F.R., S.L.S., J.M.S., A.M.T.O., A.C.W., M.A.W., S.Z., J.M.Z., E.E.E., R.J.O., M.C.S., K.H.M., K.D.M. and A.M.P. wrote the manuscript draft. A.R. and A.M.P. edited the manuscript, with the assistance of all authors. J.M.Z., E.E.E., R.J.O., M.C.S., K.H.M., K.D.M. and A.M.P. supervised the research. Conceptualization was the responsibility of A.R., S.N., M.C., E.E.E., K.H.M., K.D.M. and A.M.P.
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S.N. is now an employee of ONT. S.K. has received travel funding for speaking at events hosted by ONT. A.F. is an employee of DNAnexus. C.-S.C. is an employee of GeneDX Holdings Corp. N.-C.C. is an employee of Exai Bio. L.F.P. receives research support from Genetech. F.J.S. receives research support from Pacific Biosciences, ONT, Illumina and Genetech. K.S. is an employee of Google LLC and owns Alphabet stock as part of the standard compensation package. W.T. has two patents (nos. 8,748,091 and 8,394,584) licensed to ONT. E.E.E. is a scientific advisory board member of Variant Bio, Inc. The remaining authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Assembling the X and Y chromosomes of HG002.
a. Chromosome X and Y components of the assembly string graph built from HiFi reads, detected based on node sequence alignments to T2T-CHM13 and GRCh38 references. Each node is colored according to the excess of paternal-specific (blue) and maternal-specific (red) k-mers, obtained from parental Illumina reads, indicating if they exclusively belong to chromosome Y or X, respectively. Most complicated tangles are localized within the heterochromatic satellite region on the Y q-arm. The X and Y subgraphs are connected in PAR1 and PAR2. Graph discontinuities are due to a lack of HiFi sequence coverage in these regions caused by contextual sequencing bias, with 9 out of 11 observed breaks falling within PAR1 on either chromosome (5 out of 5 for chromosome Y). Note that for visualization purposes the length of shorter nodes is artificially increased making the extent of the tangles appear larger than reality. b. The effects of manual pruning and semi-automated ONT read integration is illustrated from top to bottom. Top, zoomed in view of a tangle encoding the P1–P3 palindromic region in Y (approx. 22.86–27.08 Mb, see Fig. 4). Middle, corresponding subgraph following the manual pruning and recompaction. Nodes excluded from the curated “single-copy” list for automated ONT-based repeat resolution are shown in yellow. Three hairpin structures are highlighted, which form almost-perfect inverted tandem repeats encompassing the entire P3 and two P2 (red) palindromes. Node outlines in the palindromes are colored according to the palindromic arms as in Fig. 4. Bottom, corresponding subgraph following the repeat resolution using ONT read-to-graph alignments. Remaining ambiguities were resolved by evaluating ONT read alignments to all candidate reconstructions of the corresponding sub-regions. c. PAR1 subgraph labeled with HiFi read coverage on each node. Gaps (green edges) and uneven node coverage estimates indicate biases in HiFi sequencing across the region. Figure 1 shows an enrichment of SINE repeats and non-B DNA motifs in PAR1 that may underlie the sequencing gaps in this region.
Extended Data Fig. 2 Validation and polishing of the T2T-Y.
a. Evaluation and polishing workflow performed on T2T-CHM13v1.1 autosomes + HG002 XY assemblies. b. Venn diagram of the k-mers from the parents and child. On the left, hap-mers18 represent haplotype specific k-mers inherited by the child. The darker outlined circle inside the child k-mers represent single-copy k-mers (k-mers occurring once in the assembly and single-copy in the child’s genome). Right figure shows an example of the paternal specific, “single-copy” and “marker” k-mers. The marker set includes both multi-copy and single-copy k-mers specific to the paternal haplotype that were inherited by the child. Unlike polishing the nearly haploid CHM13 assembly17, both single-copy k-mers and marker k-mers were used for the marker-assisted alignments to HG002 XY. This helped align more reads within repetitive regions to the correct chromosome for evaluation during polishing. Right panel shows counts of the k-mers and coverage of HiFi and ONT reads using the marker-assisted Winnowmap2 alignment, in addition to alignments from VerityMap, which uses locally unique k-mers for anchoring the reads. c. Aggregated Strand-seq coverage profile across all 65 libraries on GRCh38-Y (top) and T2T-Y (bottom). Each bar represents read counts in every 20 kb bin supporting the reference in forward direction (light green) or reverse direction (dark green). Multiple spikes in reverse direction (black asterisks) in GRCh38-Y indicate inversion polymorphisms relative to HG002, likely due to differences between the haplogroups. Such spikes in coverage are not observed on T2T-X and T2T-Y, which confirm the structural and directional accuracy of the HG002 assemblies. A 3 kb inversion of the unique sequence between the P5 palindromic arms was identified as erroneous in T2T-Y (red asterisk), but was confirmed to be polymorphic in the population and left uncorrected in this version of the assembly.
Extended Data Fig. 3 Large structural differences between T2T-Y and previous GRCh Y assemblies.
a-b. Ampliconic genes and X-degenerate sequences revealed from alignments between GRCh38-Y (Y-axis) and T2T-Y (X-axis). a. Dotplot generated using LastZ93 after softmasking with WindowMasker94. b. Identity was computed from matches and mismatches over positions with alignments, excluding gaps. c. Structural differences revealed using PRG-TK95 against GRCh38-Y and GRCh37-Y in the euchromatic region of the Y chromosome.
Extended Data Fig. 4 Repeat discovery and annotation of T2T-Y.
a. Assembly completion allowed for a full assessment of repeats and resulted in the identification of previously unknown satellite arrays (predominantly in the PAR1) and subunit repeats that fall within one of three composite repeat units (TSPY, RBMY, DAZ). b. Ideogram of TE density (per 100 kb bin). This is an extension of Fig. 1 with non-SINEs expanded into separate TE classes (SVA, LTR, LINE, DNA/RC). Density scale ranges from low (white, zero) to high (black, relative to total density) and sequence classes are denoted by color. c. Summary (in terms of base coverage per region) across all five TE classes and two specific families: Alu/SINE and L1/LINE. The satellites in (b) were kept separate as two categories; Cen/Sat as the left satellite block including alpha satellites and DYZ19, while all other categories were combined per sequence classes.
Extended Data Fig. 5 Non-B DNA motifs along the T2T-Y.
HSat3 on the Yq and satellite sequences around the centromere are more enriched with A-phased repeats, direct repeats and STRs, while HSat1B is more enriched with inverted repeats and mirror repeats. Enrichment of non-B DNA sequences were also observed in the PAR region. Notably, the TSPY gene array is enriched for G4 and Z-DNA motifs, as shown in Extended Data Fig. 6b.
Extended Data Fig. 6 Phylogenetic tree analysis of the ampliconic TSPY gene family and pattern of non-B DNA structure.
a. Phylogenetic tree analysis using protein-coding TSPYs from a Sumatran Orangutan (Pongo abelii) and a Silvery gibbon (Hylobates moloch) as outgroups confirmed TSPY2 (distal to the array) and TSPY copies within the array originated from the same branch, distinguished from the rest of the TSPY pseudogenes. Rectangular inset shows a cartoon representation of the simplified tree. Numbers next to the triangles indicate the number of TSPY genes in the same branch. b. G4 and Z-DNA structures predicted for a typical TSPY copy inside the TSPY array. All TSPY copies in the array have the same signature, with one G4 peak present ~500 bases upstream of the TSPY (arrow). Higher Quadron score122 (Q-score) indicates a more stable G4 structure, with scores over 19 considered stable (dotted line).
Extended Data Fig. 7 Recurrent inversions identified with Strand-seq.
a. Five out of 15 individuals have the inverted variant as present in HG002 at the P3 palindrome (white arrow). Although inversions across P1–P2 (yellow and red arrows) are difficult to confirm with Strand-seq because of the high sequence similarity between the palindromic arms, different orientations are observable in these samples. b. Strand states for 65 Strand-seq libraries of HG002. Depending on the mappings of directional Strand-seq reads (+ reads: ‘Crick’, C, – reads: ‘Watson’, W), reference sequence was assigned in three states: WC, WW, and CC. WC, roughly equal mixture of plus and minus reads; WW, all reads mapped in minus orientation; CC, all reads mapped in plus orientation. Changes in strand state along a single chromosome are normally caused by a double-strand-break (DSBs) that occurred during DNA replication160 in a random fashion and we refer to them as sister-chromatid-exchanges (SCEs, yellow thunderbolts). Recurrent change in strand state over the same region in multiple Strand-seq cells indicates misassembly. Similarly, collapsed or incomplete assembly of a certain genomic region will result in a recurrent strand state change as observed for GRCh38-Y (black arrowheads). In contrast, T2T-Y shows strand state changes randomly distributed along each Strand-seq library with no evidence of misassembly or collapse. c. Strand-seq profile of selected libraries over T2T-Y summarized in bins (bin size: 500 kb, step size: 50 kb). Teal, Crick read counts; orange, Watson read counts. As ChrY is haploid, reads are expected to map only in Watson or Crick orientation. Light gray rectangles highlight regions where SCEs were detected in the heterochromatic Yq12 despite a lower coverage of Strand-seq reads. A modified breakpointR parameter was used (windowsize = 500000 minReads = 20) in order to refine detected SCEs presented in panel b and c.
Extended Data Fig. 8 Satellite annotation and recent expansion events in the Yq heterochromatin.
a. A plot showing the top repeat periodicities detected by NTRprism44 in 50 kb blocks tiled across T2T-Y, with centromeric satellite annotations overlaid on the X axis. Large arrays are labeled with their historic nomenclature1, HSat subfamilies61, and predominant repeat periodicities. b. An exact 2000-mer match dotplot of the Yq region (a dot is plotted when an identical 2000 base sequence is found at positions X and Y). The lower triangle has DYZ1/DYZ2 annotations overlaid as yellow and blue bars, respectively. Circled patterns in the upper triangle correspond to recent iterative duplication events, which are illustrated below the X axis. c. A reconstruction of a possible sequence of recent iterative duplications that could explain the observed dotplot patterns. d. A 2000-mer dotplot comparison of two ~800 kb HSat1B sub-arrays that were part of a recent large duplication event, along with self-self comparisons of the same arrays, revealing sites of more recent and smaller-scale deletions and expansions (annotated in yellow and red, with a possible sequence of events illustrated by the schematic on the right).
Extended Data Fig. 9 Genomic similarity in PARs and XTR and improved MAPQ of the PARs through informed sex chromosome complement reference.
a. Dotplots from LASTZ alignments of the CHM13-X, HG002-X, and HG002-Y (T2T-Y) over 96% sequence identity. Dashed gray lines represent the start and end of the approximate PARs or XTR boundaries. Disconnected diagonal lines indicate the presence of genomic diversity between each paired region. More genomic differences are observed in the PAR1 between the HG002-Y and CHM13-X. b-c. Average mapping quality (MAPQ) across GRCh38-X from simulated reads of an XX (b) and XY (c) sample. Top, a default version of GRCh38 (with two copies of identical PARs on XY). Middle, a version of GRCh38 informed on the sex chromosome complement (SCC) of the sample (entire Y hard-masked for the XX sample vs. only PARs on the Y hard-masked for the XY sample). Bottom, the difference in average MAPQ between the SCC and default approaches. MAPQ was averaged in 50 kb windows, sliding 10 kb across the chromosome. A positive value means MAPQ score is higher with SCC reference alignment compared to default alignment.
Extended Data Fig. 10 Number of variants called from 1KGP and SGDP individuals.
a. More variants are called on the X-PARs when using the sex chromosome complement reference approach (calling variants in diploid mode on PARs) than the non-masked approach (calling variants in haploid mode on PARs). The 1KGP results for GRCh38-Y are from Aganezov et al.66, which was performed on CHM13v1.0+GRCh38-Y. b. Num. of variants called from each 1KGP XY sample on chromosome GRCh38-Y and T2T-Y c. Num. of variants called in the syntenic region between the two Ys. A large num. of additional variants are called on each sample attributed to the newly added, non-syntenic sequences on T2T-Y. Within the syntenic regions, a reduction in the number of variants is observed for each population except for samples from R1 haplogroups as shown in Fig. 6c. d. Aggregated total number of variants for the 279 SGDP samples per chromosome. e. SGDP genome-wide counts of variants per-sample (n = 279) demonstrate increased variation in African samples regardless of reference. Each bar in the box plot represents the 1st, 2nd (median), and 3rd quartile of the number of variants in each population. Whiskers are bound to the 1.5 × interquartile range. Data outside of the whisker ranges are shown as dots. For the SGDP samples, variants were called using T2T-CHM13+Y or GRCh38 as the reference. All variants shown in this figure were filtered for “high quality (PASS)”.
Extended Data Fig. 11 Human contaminants in bacterial reference genomes.
a. Number of distinct RefSeq accessions in every 10 kb window containing 64-mers of GRCh38-Y (top), T2T-Y (middle), and in T2T-Y only (bottom). Here, RefSeq sequences with more than 20 64-mers or matching over 10% of the Y chromosome are included. b. Length distribution of the sequences from (a) in log scale. Majority of the shorter (<1 kb) sequences contain 64-mers found in HSat1B or HSat3. c. Number of bacterial RefSeq entries by strain identified to contain sequences of T2T-Y and not GRCh38-Y, visualized with Krona158.
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Rhie, A., Nurk, S., Cechova, M. et al. The complete sequence of a human Y chromosome. Nature 621, 344–354 (2023). https://doi.org/10.1038/s41586-023-06457-y
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DOI: https://doi.org/10.1038/s41586-023-06457-y
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