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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.

There has been tremendous progress in phased genome assembly production by combining long-read data with parental information or linked-read data. Nevertheless, a typical phased genome assembly generated by trio-hifiasm still generates more than 140 gaps. We perform a detailed analysis of gaps, assembly breaks, and misorientations from 182 haploid assemblies obtained from a diversity panel of 77 unique human samples. Although trio-based approaches using HiFi are the current gold standard, chromosome-wide phasing accuracy is comparable when using Strand-seq instead of parental data. Importantly, the majority of assembly gaps cluster near the largest and most identical repeats (including segmental duplications [35.4%], satellite DNA [22.3%], or regions enriched in GA/AT-rich DNA [27.4%]). Consequently, 1513 protein-coding genes overlap assembly gaps in at least one haplotype, and 231 are recurrently disrupted or missing from five or more haplotypes. Furthermore, we estimate that 6-7 Mbp of DNA are misorientated per haplotype irrespective of whether trio-free or trio-based approaches are used. Of these misorientations, 81% correspond to bona fide large inversion polymorphisms in the human species, most of which are flanked by large segmental duplications. We also identify large-scale alignment discontinuities consistent with 11.9 Mbp of deletions and 161.4 Mbp of insertions per haploid genome. Although 99% of this variation corresponds to satellite DNA, we identify 230 regions of euchromatic DNA with frequent expansions and contractions, nearly half of which overlap with 197 protein-coding genes. Such variable and incompletely assembled regions are important targets for future algorithmic development and pangenome representation.

Genome in a Bottle benchmarks are widely used to help validate clinical sequencing pipelines and develop variant calling and sequencing methods. Here we use accurate linked and long reads to expand benchmarks in 7 samples to include difficult-to-map regions and segmental duplications that are challenging for short reads. These benchmarks add more than 300,000 SNVs and 50,000 insertions or deletions (indels) and include 16% more exonic variants, many in challenging, clinically relevant genes not covered previously, such as PMS2. For HG002, we include 92% of the autosomal GRCh38 assembly while excluding regions problematic for benchmarking small variants, such as copy number variants, that should not have been in the previous version, which included 85% of GRCh38. It identifies eight times more false negatives in a short read variant call set relative to our previous benchmark. We demonstrate that this benchmark reliably identifies false positives and false negatives across technologies, enabling ongoing methods development.

Methods that use a linear genome reference for genome sequencing data analysis are reference-biased. In the field of clinical genetics for rare diseases, a resulting reduction in genotyping accuracy in some regions has likely prevented the resolution of some cases. Pangenome graphs embed population variation into a reference structure. Although pangenome graphs have helped to reduce reference mapping bias, further performance improvements are possible. We introduce VG-Pedigree, a pedigree-aware workflow based on the pangenome-mapping tool of Giraffe and the variant calling tool DeepTrio using a specially trained model for Giraffe-based alignments. We demonstrate mapping and variant calling improvements in both single-nucleotide variants (SNVs) and insertion and deletion (indel) variants over those produced by alignments created using BWA-MEM to a linear-reference and Giraffe mapping to a pangenome graph containing data from the 1000 Genomes Project. We have also adapted and upgraded deleterious-variant (DV) detecting methods and programs into a streamlined workflow. We used these workflows in combination to detect small lists of candidate DVs among 15 family quartets and quintets of the Undiagnosed Diseases Program (UDP). All candidate DVs that were previously diagnosed using the Mendelian models covered by the previously published methods were recapitulated by these workflows. The results of these experiments indicate that a slightly greater absolute count of DVs are detected in the proband population than in their matched unaffected siblings.

More than 40% of the germline variants in ClinVar today are variants of uncertain significance (VUSs). These variants remain unclassified in part because the patient-level data needed for their interpretation is siloed. Federated analysis can overcome this problem by "bringing the code to the data": analyzing the sensitive patient-level data computationally within its secure home institution and providing researchers with valuable insights from data that would not otherwise be accessible. We tested this principle with a federated analysis of breast cancer clinical data at RIKEN, derived from the BioBank Japan repository. We were able to analyze these data within RIKEN's secure computational framework without the need to transfer the data, gathering evidence for the interpretation of several variants. This exercise represents an approach to help realize the core charter of the Global Alliance for Genomics and Health (GA4GH): to responsibly share genomic data for the benefit of human health.

We introduce Giraffe, a pangenome short-read mapper that can efficiently map to a collection of haplotypes threaded through a sequence graph. Giraffe maps sequencing reads to thousands of human genomes at a speed comparable to that of standard methods mapping to a single reference genome. The increased mapping accuracy enables downstream improvements in genome-wide genotyping pipelines for both small variants and larger structural variants. We used Giraffe to genotype 167,000 structural variants, discovered in long-read studies, in 5202 diverse human genomes that were sequenced using short reads. We conclude that pangenomics facilitates a more comprehensive characterization of variation and, as a result, has the potential to improve many genomic analyses.

ABSTRACT We introduce Giraffe, a pangenome short read mapper that can efficiently map to a collection of haplotypes threaded through a sequence graph. Giraffe, part of the variation graph toolkit (vg) 1 , maps reads to thousands of human genomes at around the same speed BWA-MEM 2 maps reads to a single reference genome, while maintaining comparable accuracy to VG-MAP, vg’s original mapper. We have developed efficient genotyping pipelines using Giraffe. We demonstrate improvements in genotyping for single-nucleotide variants (SNVs), small insertions and deletions (indels) and structural variations (SVs) genome-wide. We use Giraffe to genotype about 167 thousand structural variants ascertained from long read studies in 5,202 human genomes sequenced with short reads, including the complete 1000 Genomes Project dataset, at an average cost of $1.50 per sample. We determine the frequency of these variations in diverse human populations, characterize their complex allelic variations and identify thousands of expression quantitative trait loci (eQTLs) driven by these variations.

In March 2019, 45 scientists and software engineers from around the world converged at the University of California, Santa Cruz for the first pangenomics codeathon. The purpose of the meeting was to propose technical specifications and standards for a usable human pangenome as well as to build relevant tools for genome graph infrastructures. During the meeting, the group held several intense and productive discussions covering a diverse set of topics, including advantages of graph genomes over a linear reference representation, design of new methods that can leverage graph-based data structures, and novel visualization and annotation approaches for pangenomes. Additionally, the participants self-organized themselves into teams that worked intensely over a three-day period to build a set of pipelines and tools for specific pangenomic applications. A summary of the questions raised and the tools developed are reported in this manuscript.

The BRCA Challenge is a long-term data-sharing project initiated within the Global Alliance for Genomics and Health (GA4GH) to aggregate BRCA1 and BRCA2 data to support highly collaborative research activities. Its goal is to generate an informed and current understanding of the impact of genetic variation on cancer risk across the iconic cancer predisposition genes, BRCA1 and BRCA2. Initially, reported variants in BRCA1 and BRCA2 available from public databases were integrated into a single, newly created site, www.brcaexchange.org. The purpose of the BRCA Exchange is to provide the community with a reliable and easily accessible record of variants interpreted for a high-penetrance phenotype. More than 20,000 variants have been aggregated, three times the number found in the next-largest public database at the project's outset, of which approximately 7,250 have expert classifications. The data set is based on shared information from existing clinical databases-Breast Cancer Information Core (BIC), ClinVar, and the Leiden Open Variation Database (LOVD)-as well as population databases, all linked to a single point of access. The BRCA Challenge has brought together the existing international Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium expert panel, along with expert clinicians, diagnosticians, researchers, and database providers, all with a common goal of advancing our understanding of BRCA1 and BRCA2 variation. Ongoing work includes direct contact with national centers with access to BRCA1 and BRCA2 diagnostic data to encourage data sharing, development of methods suitable for extraction of genetic variation at the level of individual laboratory reports, and engagement with participant communities to enable a more comprehensive understanding of the clinical significance of genetic variation in BRCA1 and BRCA2.

Reference genomes guide our interpretation of DNA sequence data. However, conventional linear references represent only one version of each locus, ignoring variation in the population. Poor representation of an individual's genome sequence impacts read mapping and introduces bias. Variation graphs are bidirected DNA sequence graphs that compactly represent genetic variation across a population, including large-scale structural variation such as inversions and duplications. Previous graph genome software implementations have been limited by scalability or topological constraints. Here we present vg, a toolkit of computational methods for creating, manipulating, and using these structures as references at the scale of the human genome. vg provides an efficient approach to mapping reads onto arbitrary variation graphs using generalized compressed suffix arrays, with improved accuracy over alignment to a linear reference, and effectively removing reference bias. These capabilities make using variation graphs as references for DNA sequencing practical at a gigabase scale, or at the topological complexity of de novo assemblies.