Scalable telomere-to-telomere assembly for diploid and polyploid genomes with double graph.

Despite advances in long-read sequencing technologies, constructing a near telomere-to-telomere assembly is still computationally demanding. Here we present hifiasm (UL), an efficient de novo assembly algorithm combining multiple sequencing technologies to scale up population-wide near telomere-to-telomere assemblies. Applied to 22 human and two plant genomes, our algorithm produces better diploid assemblies at a cost of an order of magnitude lower than existing methods, and it also works with polyploid genomes.

Phased nanopore assembly with Shasta and modular graph phasing with GFAse.

Reference-free genome phasing is vital for understanding allele inheritance and the impact of single-molecule DNA variation on phenotypes. To achieve thorough phasing across homozygous or repetitive regions of the genome, long-read sequencing technologies are often used to perform phased de novo assembly. As a step toward reducing the cost and complexity of this type of analysis, we describe new methods for accurately phasing Oxford Nanopore Technologies (ONT) sequence data with the Shasta genome assembler and a modular tool for extending phasing to the chromosome scale called GFAse. We test using new variants of ONT PromethION sequencing, including those using proximity ligation, and show that newer, higher accuracy ONT reads substantially improve assembly quality.

Scalable Nanopore sequencing of human genomes provides a comprehensive view of haplotype-resolved variation and methylation.

Long-read sequencing technologies substantially overcome the limitations of short-reads but have not been considered as a feasible replacement for population-scale projects, being a combination of too expensive, not scalable enough or too error-prone. Here we develop an efficient and scalable wet lab and computational protocol, Napu, for Oxford Nanopore Technologies long-read sequencing that seeks to address those limitations. We applied our protocol to cell lines and brain tissue samples as part of a pilot project for the National Institutes of Health Center for Alzheimer's and Related Dementias. Using a single PromethION flow cell, we can detect single nucleotide polymorphisms with F1-score comparable to Illumina short-read sequencing. Small indel calling remains difficult within homopolymers and tandem repeats, but achieves good concordance to Illumina indel calls elsewhere. Further, we can discover structural variants with F1-score on par with state-of-the-art de novo assembly methods. Our protocol phases small and structural variants at megabase scales and produces highly accurate, haplotype-specific methylation calls.

The complete sequence of a human Y chromosome.

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 duplications1-3. 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.

Scalable telomere-to-telomere assembly for diploid and polyploid genomes with double graph.

Despite recent advances in the length and the accuracy of long-read data, building haplotype-resolved genome assemblies from telomere to telomere still requires considerable computational resources. In this study, we present an efficient de novo assembly algorithm that combines multiple sequencing technologies to scale up population-wide telomere-to-telomere assemblies. By utilizing twenty-two human and two plant genomes, we demonstrate that our algorithm is around an order of magnitude cheaper than existing methods, while producing better diploid and haploid assemblies. Notably, our algorithm is the only feasible solution to the haplotype-resolved assembly of polyploid genomes.

Increased mutation and gene conversion within human segmental duplications.

Single-nucleotide variants (SNVs) in segmental duplications (SDs) have not been systematically assessed because of the limitations of mapping short-read sequencing data1,2. Here we constructed 1:1 unambiguous alignments spanning high-identity SDs across 102 human haplotypes and compared the pattern of SNVs between unique and duplicated regions3,4. We find that human SNVs are elevated 60% in SDs compared to unique regions and estimate that at least 23% of this increase is due to interlocus gene conversion (IGC) with up to 4.3 megabase pairs of SD sequence converted on average per human haplotype. We develop a genome-wide map of IGC donors and acceptors, including 498 acceptor and 454 donor hotspots affecting the exons of about 800 protein-coding genes. These include 171 genes that have 'relocated' on average 1.61 megabase pairs in a subset of human haplotypes. Using a coalescent framework, we show that SD regions are slightly evolutionarily older when compared to unique sequences, probably owing to IGC. SNVs in SDs, however, show a distinct mutational spectrum: a 27.1% increase in transversions that convert cytosine to guanine or the reverse across all triplet contexts and a 7.6% reduction in the frequency of CpG-associated mutations when compared to unique DNA. We reason that these distinct mutational properties help to maintain an overall higher GC content of SD DNA compared to that of unique DNA, probably driven by GC-biased conversion between paralogous sequences5,6.

A draft human pangenome reference.

Here the Human Pangenome Reference Consortium presents a first draft of the human pangenome reference. The pangenome contains 47 phased, diploid assemblies from a cohort of genetically diverse individuals1. These assemblies cover more than 99% of the expected sequence in each genome and are more than 99% accurate at the structural and base pair levels. Based on alignments of the assemblies, we generate a draft pangenome that captures known variants and haplotypes and reveals new alleles at structurally complex loci. We also add 119 million base pairs of euchromatic polymorphic sequences and 1,115 gene duplications relative to the existing reference GRCh38. Roughly 90 million of the additional base pairs are derived from structural variation. Using our draft pangenome to analyse short-read data reduced small variant discovery errors by 34% and increased the number of structural variants detected per haplotype by 104% compared with GRCh38-based workflows, which enabled the typing of the vast majority of structural variant alleles per sample.

Phased nanopore assembly with Shasta and modular graph phasing with GFAse.

As a step towards simplifying and reducing the cost of haplotype resolved de novo assembly, we describe new methods for accurately phasing nanopore data with the Shasta genome assembler and a modular tool for extending phasing to the chromosome scale called GFAse. We test using new variants of Oxford Nanopore Technologies' (ONT) PromethION sequencing, including those using proximity ligation and show that newer, higher accuracy ONT reads substantially improve assembly quality.

Scalable Nanopore sequencing of human genomes provides a comprehensive view of haplotype-resolved variation and methylation.

Long-read sequencing technologies substantially overcome the limitations of short-reads but to date have not been considered as feasible replacement at scale due to a combination of being too expensive, not scalable enough, or too error-prone. Here, we develop an efficient and scalable wet lab and computational protocol for Oxford Nanopore Technologies (ONT) long-read sequencing that seeks to provide a genuine alternative to short-reads for large-scale genomics projects. We applied our protocol to cell lines and brain tissue samples as part of a pilot project for the NIH Center for Alzheimer's and Related Dementias (CARD). Using a single PromethION flow cell, we can detect SNPs with F1-score better than Illumina short-read sequencing. Small indel calling remains to be difficult inside homopolymers and tandem repeats, but is comparable to Illumina calls elsewhere. Further, we can discover structural variants with F1-score comparable to state-of the-art methods involving Pacific Biosciences HiFi sequencing and trio information (but at a lower cost and greater throughput). Using ONT based phasing, we can then combine and phase small and structural variants at megabase scales. Our protocol also produces highly accurate, haplotype-specific methylation calls. Overall, this makes large-scale long-read sequencing projects feasible; the protocol is currently being used to sequence thousands of brain-based genomes as a part of the NIH CARD initiative. We provide the protocol and software as open-source integrated pipelines for generating phased variant calls and assemblies.

Semi-automated assembly of high-quality diploid human reference genomes.

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.

The Human Pangenome Project: a global resource to map genomic diversity.

The human reference genome is the most widely used resource in human genetics and is due for a major update. Its current structure is a linear composite of merged haplotypes from more than 20 people, with a single individual comprising most of the sequence. It contains biases and errors within a framework that does not represent global human genomic variation. A high-quality reference with global representation of common variants, including single-nucleotide variants, structural variants and functional elements, is needed. The Human Pangenome Reference Consortium aims to create a more sophisticated and complete human reference genome with a graph-based, telomere-to-telomere representation of global genomic diversity. Here we leverage innovations in technology, study design and global partnerships with the goal of constructing the highest-possible quality human pangenome reference. Our goal is to improve data representation and streamline analyses to enable routine assembly of complete diploid genomes. With attention to ethical frameworks, the human pangenome reference will contain a more accurate and diverse representation of global genomic variation, improve gene-disease association studies across populations, expand the scope of genomics research to the most repetitive and polymorphic regions of the genome, and serve as the ultimate genetic resource for future biomedical research and precision medicine.

Complete genomic and epigenetic maps of human centromeres.

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.