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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.

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.

Diverse sets of complete human genomes are required to construct a pangenome reference and to understand the extent of complex structural variation. Here, we sequence 65 diverse human genomes and build 130 haplotype-resolved assemblies (130 Mbp median continuity), closing 92% of all previous assembly gaps 1,2 and reaching telomere-to-telomere (T2T) status for 39% of the chromosomes. We highlight complete sequence continuity of complex loci, including the major histocompatibility complex (MHC), SMN1 / SMN2 , NBPF8 , and AMY1/AMY2 , and fully resolve 1,852 complex structural variants (SVs). In addition, we completely assemble and validate 1,246 human centromeres. We find up to 30-fold variation in α-satellite high-order repeat (HOR) array length and characterize the pattern of mobile element insertions into α-satellite HOR arrays. While most centromeres predict a single site of kinetochore attachment, epigenetic analysis suggests the presence of two hypomethylated regions for 7% of centromeres. Combining our data with the draft pangenome reference 1 significantly enhances genotyping accuracy from short-read data, enabling whole-genome inference 3 to a median quality value (QV) of 45. Using this approach, 26,115 SVs per sample are detected, substantially increasing the number of SVs now amenable to downstream disease association studies., Competing Interests: Competing Interests E.E.E. is a scientific advisory board member of Variant Bio, Inc. C. Lee is a scientific advisory board member of Nabsys and Genome Insight. S.K. has received travel funds to speak at events hosted by Oxford Nanopore Technologies. The following authors have previously disclosed a patent application (No. EP19169090) relevant to Strand-seq: J.O.K., T.M., and D.P. The other authors declare no competing interests.

Background: The duplication-triplication/inverted-duplication (DUP-TRP/INV-DUP) structure is a type of complex genomic rearrangement (CGR) hypothesized to result from replicative repair of DNA due to replication fork collapse. It is often mediated by a pair of inverted low-copy repeats (LCR) followed by iterative template switches resulting in at least two breakpoint junctions in cis . Although it has been identified as an important mutation signature of pathogenicity for genomic disorders and cancer genomes, its architecture remains unresolved and is predicted to display at least four structural variation (SV) haplotypes., Results: Here we studied the genomic architecture of DUP-TRP/INV-DUP by investigating the genomic DNA of 24 patients with neurodevelopmental disorders identified by array comparative genomic hybridization (aCGH) on whom we found evidence for the existence of 4 out of 4 predicted SV haplotypes. Using a combination of short-read genome sequencing (GS), long- read GS, optical genome mapping and StrandSeq the haplotype structure was resolved in 18 samples. This approach refined the point of template switching between inverted LCRs in 4 samples revealing a DNA segment of ∼2.2-5.5 kb of 100% nucleotide similarity. A prediction model was developed to infer the LCR used to mediate the non-allelic homology repair., Conclusions: These data provide experimental evidence supporting the hypothesis that inverted LCRs act as a recombinant substrate in replication-based repair mechanisms. Such inverted repeats are particularly relevant for formation of copy-number associated inversions, including the DUP-TRP/INV-DUP structures. Moreover, this type of CGR can result in multiple conformers which contributes to generate diverse SV haplotypes in susceptible loci .