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.

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.

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.

In 2001, Celera Genomics and the International Human Genome Sequencing Consortium published their initial drafts of the human genome, which revolutionized the field of genomics. While these drafts and the updates that followed effectively covered the euchromatic fraction of the genome, the heterochromatin and many other complex regions were left unfinished or erroneous. Addressing this remaining 8% of the genome, the Telomere-to-Telomere (T2T) Consortium has finished the first truly complete 3.055 billion base pair (bp) sequence of a human genome, representing the largest improvement to the human reference genome since its initial release. The new T2T-CHM13 reference includes gapless assemblies for all 22 autosomes plus chromosome X, corrects numerous errors, and introduces nearly 200 million bp of novel sequence containing 2,226 paralogous gene copies, 115 of which are predicted to be protein coding. The newly completed regions include all centromeric satellite arrays and the short arms of all five acrocentric chromosomes, unlocking these complex regions of the genome to variational and functional studies for the first time.

Long-read sequencing has the potential to transform variant detection by reaching currently difficult-to-map regions and routinely linking together adjacent variations to enable read based phasing. Third-generation nanopore sequence data has demonstrated a long read length, but current interpretation methods for its novel pore-based signal have unique error profiles, making accurate analysis challenging. Here, we introduce a haplotype-aware variant calling pipeline PEPPER-Margin-DeepVariant that produces state-of-the-art variant calling results with nanopore data. We show that our nanopore-based method outperforms the short-read-based single nucleotide variant identification method at the whole genome-scale and produces high quality single nucleotide variants in segmental duplications and low-mappability regions where short-read based genotyping fails. We show that our pipeline can provide highly-contiguous phase blocks across the genome with nanopore reads, contiguously spanning between 85% to 92% of annotated genes across six samples. We also extend PEPPER-Margin-DeepVariant to PacBio HiFi data, providing an efficient solution with superior performance than the current WhatsHap-DeepVariant standard. Finally, we demonstrate de novo assembly polishing methods that use nanopore and PacBio HiFi reads to produce diploid assemblies with high accuracy (Q35+ nanopore-polished and Q40+ PacBio-HiFi-polished).

Genome in a Bottle (GIAB) benchmarks have been widely used to validate clinical sequencing pipelines and develop new variant calling and sequencing methods. Here we use accurate long and linked reads to expand the prior benchmark to include difficult-to-map regions and segmental duplications that are not readily accessible to short reads. Our new benchmark adds more than 300,000 SNVs, 50,000 indels, and 16 % new exonic variants, many in challenging, clinically relevant genes not previously covered (e.g., PMS2). We increase coverage of the GRCh38 assembly from 85 % to 92 %, while excluding problematic regions for benchmarking small variants (e.g., copy number variants and assembly errors) that should not have been in the previous version. Our new benchmark reliably identifies both false positives and false negatives across multiple short-, linked-, and long-read based variant calling methods. As an example of its utility, this benchmark identifies eight times more false negatives in a short read variant call set relative to our previous benchmark, mostly in difficult-to-map regions. To enable robust small variant benchmarking, we still exclude 3.6% of GRCh37 and 5.0% of GRCh38 in (1) highly repetitive regions such as large, highly similar segmental duplications and the centromere not accessible to our data and (2) regions where our sample is highly divergent from the reference due to large indels, structural variation, copy number variation, and/or errors in the reference (e.g., some KIR genes that have duplications in HG002). We have demonstrated the utility of this benchmark to assess performance in more challenging regions, which enables benchmarking in more difficult genes and continued technology and bioinformatics development. The benchmarks are available at: ftp://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/release/AshkenazimTrio/HG002_NA24385_son/NISTv4.1/ftp://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/AshkenazimTrio/analysis/NIST_v4.2_SmallVariantDraftBenchmark_07092020/

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The current human reference genome, GRCh38, represents over 20 years of effort to generate a high-quality assembly, which has benefitted society1,2. However, it still has many gaps and errors, and does not represent a biological genome as it is a blend of multiple individuals3,4. Recently, a high-quality telomere-to-telomere reference, CHM13, was generated with the latest long-read technologies, but it was derived from a hydatidiform mole cell line with a nearly homozygous genome5. To address these limitations, the Human Pangenome Reference Consortium formed with the goal of creating high-quality, cost-effective, diploid genome assemblies for a pangenome reference that represents human genetic diversity6. Here, in our first scientific report, we determined which combination of current genome sequencing and assembly approaches yield the most complete and accurate diploid genome assembly with minimal manual curation. Approaches that used highly accurate long reads and parent–child data with graph-based haplotype phasing during assembly outperformed those that did not. Developing a combination of the top-performing methods, we generated our first high-quality diploid reference assembly, containing only approximately four gaps per chromosome on average, with most chromosomes within ±1% of the length of CHM13. Nearly 48% of protein-coding genes have non-synonymous amino acid changes between haplotypes, and centromeric regions showed the highest diversity. Our findings serve as a foundation for assembling near-complete diploid human genomes at scale for a pangenome reference to capture global genetic variation from single nucleotides to structural rearrangements.