Telomere-to-telomere assembly of a complete human X chromosome

Abstract

After two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no single chromosome has been finished end to end, and hundreds of unresolved gaps persist 1,2. Here we present a human genome assembly that surpasses the continuity of GRCh38 2 , along with a gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome 3 , we reconstructed the centromeric satellite DNA array (approximately 3.1 Mb) and closed the 29 remaining gaps in the current reference, including new sequences from the human pseudoautosomal regions and from cancer-testis ampliconic gene families (CT-X and GAGE). These sequences will be integrated into future human reference genome releases. In addition, the complete chromosome X, combined with the ultra-long nanopore data, allowed us to map methylation patterns across complex tandem repeats and satellite arrays. Our results demonstrate that finishing the entire human genome is now within reach, and the data presented here will facilitate ongoing efforts to complete the other human chromosomes. Complete, telomere-to-telomere reference genome assemblies are necessary to ensure that all genomic variants are discovered and studied. At present, unresolved areas of the human genome are defined by multi-megabase satellite arrays in the pericentromeric regions and the ribosomal DNA arrays on acrocentric short arms, as well as regions enriched in segmental duplications that are greater than hundreds of kilobases in length and that exhibit sequence identity of more than 98% between paralogues. Owing to their absence from the reference, these repeat-rich sequences are often excluded from genetics and genomics studies, which limits the scope of association and functional analyses 4,5. Unresolved repeat sequences also result in unintended consequences; for example, paralogous sequence variants incorrectly being called as allelic variants 6 , and the contamination of bacterial gene databases 7. Completion of the entire human genome is expected to contribute to our understanding of chromosome function 8 , human disease 9 and genomic variation, which will improve technologies in biomedicine that use short-read mapping to a reference genome (for example, RNA sequencing (RNA-seq) 10 , chromatin immunoprecipitation followed by sequencing (ChIP-seq) 11 and assay for transposase-accessible chromatin using sequencing (ATAC-seq) 12). The fundamental challenge of reconstructing a genome from many comparatively short sequencing reads-a process known as genome assembly-is distinguishing the repeated sequences from one another 13. Resolving such repeats relies on sequencing reads that are long enough to span the entire repeat or accurate enough to distinguish each repeat copy on the basis of...

HiNT: a computational method for detecting copy number variations and translocations from Hi-C data

“…Hi-C data for HelaS3, LNCaP, PANC-1, Caki2, and T47D, which were generated by the Dekker lab [38], were downloaded from the ENCODE website. Hi-C data in MCF7 and CHM13 were downloaded from GEO (GSM1631185) and the Telomere-to-Telomere consortium [39], respectively (see details in Additional file 2: Table S1).…”

Improved contiguity of the threespine stickleback genome using long-read sequencing

“…It therefore remains unknown how centromere length varies throughout the threespine stickleback genome. Complete characterization of the centromeric repetitive arrays will be aided by future sequencing of ultra-long reads (Jain et al 2018b;Miga et al 2019).…”

“…De novo assemblies of highly repetitive Y chromosomes have also become feasible using long-read sequencing (Mahajan et al 2018;Peichel et al 2019). Overall, long-read sequencing has enabled telomere-to-telomere chromosome assemblies in multiple species (Liu et al 2020;Miga et al 2019). It is clear that hybrid assembly approaches incorporating long-read sequencing have greatly improved contiguity of genomes.…”

Significantly improving the quality of genome assemblies through curation

“…Despite the advances in sequencing and mapping technologies and the ever-increasing number of sophisticated algorithms and pipelines available, generating error-free eukaryotic genome assemblies in a purely automated fashion is currently not possible [1,2]. Assembly software designed to generate continuous sequence from raw reads is confused by heterozygous or repeat-rich regions, introducing erroneous duplications, collapses and misjoins.…”