Since its initial release in 2000, the human reference genome has covered only the euchromatic fraction of the genome, leaving important heterochromatic regions unfinished. Addressing the remaining 8% of the genome, the Telomere-to-Telomere (T2T) Consortium presents a complete 3.055 billion–base pair sequence of a human genome, T2T-CHM13, that includes gapless assemblies for all chromosomes except Y, corrects errors in the prior references, and introduces nearly 200 million base pairs of sequence containing 1956 gene predictions, 99 of which are predicted to be protein coding. The completed regions include all centromeric satellite arrays, recent segmental duplications, and the short arms of all five acrocentric chromosomes, unlocking these complex regions of the genome to variational and functional studies.

The Oxford Nanopore MinION sequences individual DNA molecules using an array of pores that read nucleotide identities based on ionic current steps. We evaluated and optimized MinION performance using M13 genomic dsDNA. Using expectation-maximization (EM) we obtained robust maximum likelihood (ML) estimates for read insertion, deletion and substitution error rates (4.9%, 7.8%, and 5.1% respectively). We found that 99% of high-quality ‘2D’ MinION reads mapped to reference at a mean identity of 85%. We present a MinION-tailored tool for single nucleotide variant (SNV) detection that uses ML parameter estimates and marginalization over many possible read alignments to achieve precision and recall of up to 99%. By pairing our high-confidence alignment strategy with long MinION reads, we resolved the copy number for a cancer/testis gene family (CT47) within an unresolved region of human chromosome Xq24.

De novo assembly of a human genome using nanopore long-read sequences has been reported but it used more than 150,000 CPU hours and weeks of wall-clock time. To enable rapid human genome assembly we present Shasta, a de novo long read assembler, and polishing algorithms named MarginPolish and HELEN. Using a single PromethION nanopore sequencer and our toolkit, we assembled eleven highly contiguous human genomes de novo in nine days. We achieved ~63x coverage, 42 Kb read N50, and 6.5x coverage in 100 Kb+ reads using three flow cells per sample. Shasta produced a complete haploid human genome assembly in under six hours on a single commercial compute node. MarginPolish and HELEN polished haploid assemblies to more than 99.9% identity (QV30) with nanopore reads alone. Addition of proximity ligation (Hi-C) sequencing enabled near chromosome-level scaffolds for all eleven genomes. We compare our assembly performance to existing methods for diploid, haploid, and trio-binned human samples and report superior accuracy and speed.

Complete and accurate genome assemblies form the basis of most downstream genomic analyses and are of critical importance. Recent genome assembly projects have relied on a combination of noisy long-read sequencing and accurate shortread sequencing, with the former offering greater assembly continuity and the latter providing higher consensus accuracy. The recently introduced Pacific Biosciences (PacBio) HiFi sequencing technology bridges this divide by delivering long reads (>10 kbp) with high per-base accuracy (>99.9%). Here we present HiCanu, a modification of the Canu assembler designed to leverage the full potential of HiFi reads via homopolymer compression, overlap-based error correction, and aggressive false overlap filtering. We benchmark HiCanu with a focus on the recovery of haplotype diversity, major histocompatibility complex (MHC) variants, satellite DNAs, and segmental duplications. For diploid human genomes sequenced to 30× HiFi coverage, HiCanu achieved superior accuracy and allele recovery compared to the current state of the art. On the effectively haploid CHM13 human cell line, HiCanu achieved an NG50 contig size of 77 Mbp with a per-base consensus accuracy of 99.999% (QV50), surpassing recent assemblies of high-coverage, ultralong Oxford Nanopore Technologies (ONT) reads in terms of both accuracy and continuity. This HiCanu assembly correctly resolves 337 out of 341 validation BACs sampled from known segmental duplications and provides the first preliminary assemblies of nine complete human centromeric regions. Although gaps and errors still remain within the most challenging regions of the genome, these results represent a significant advance toward the complete assembly of human genomes.

Existing human genome assemblies have almost entirely excluded repetitive sequences within and near centromeres, limiting our understanding of their organization, evolution, and functions, which include facilitating proper chromosome segregation. Now, a complete, telomere-to-telomere human genome assembly (T2T-CHM13) has enabled us to comprehensively characterize pericentromeric and centromeric repeats, which constitute 6.2% of the genome (189.9 megabases). Detailed maps of these regions revealed multimegabase structural rearrangements, including in active centromeric repeat arrays. Analysis of centromere-associated sequences uncovered a strong relationship between the position of the centromere and the evolution of the surrounding DNA through layered repeat expansions. Furthermore, comparisons of chromosome X centromeres across a diverse panel of individuals illuminated high degrees of structural, epigenetic, and sequence variation in these complex and rapidly evolving regions.