William T. Harvey

Genetic introgression from Neanderthals and Denisovans shaped modern human genomes; however, introgressed structural variants (SVs ≥ 50 base pairs) remain challenging to discover. We integrated high-quality phased assemblies from four new Papua New Guinea (PNG) haploid genomes with 94 published assemblies of diverse ancestry to infer an introgressed SV map. Introgressed SVs are enriched in genes (47%), including critical genomic disorder regions, and are most abundant in PNG genomes. We identified 11 centromeres likely derived from archaic hominins, adding unexplored diversity to centromere genomics. Pangenome genotyping of these 98 assemblies across 1363 samples revealed 16 adaptive SVs, many associated with immune-related genes and expression, in the PNG genomes. We hypothesize that archaic SVs contributed to reproductive success, underscoring introgression as a major force in human adaptive evolution.

Chromosome 22q11.2 microdeletion syndrome (22q11.2DS) is mediated by high-identity polymorphic low-copy repeats (LCRA-to-D) that have been challenging to sequence characterize. We sequence-resolved 135 chromosome 22q11.2 haplotypes from diverse humans and define 63 distinct structural configurations differing in size by 11-fold for LCRA. This diversity is driven by a 105 kbp segmental duplication flanked by 25 kbp inverted repeats that arose in the apes but expanded in humans ~1 million years ago. African LCRA haplotypes are significantly longer ( p = 0.0047) and predicted to be more protective against 22q11.2DS ( p = 1.14×10 ^-6 ) due to enrichment of inverted 105 kbp repeats. We identify nine distinct (including five recurrent) inversions spanning LCRA-D. Sequencing four families indicates LCRA-D deletions map to 105 kbp repeats, whereas inversions map to the 25 kbp repeats. Here, we show specific haplotype LCR architectures and recurrent large-scale inversions modulate susceptibility to 22q11.2DS and help explain its reduced prevalence among individuals of African ancestry.

To better understand large-effect pathogenic variation associated with autism, we generated long-read sequencing (LRS) data to construct phased and near-complete genome assemblies (average contig N50 = 43 Mbp, QV = 56) for 189 individuals from 51 families with unsolved cases. We applied read- and assembly-based strategies to facilitate comprehensive characterization of de novo mutations, structural variants (SVs), and DNA methylation. Using LRS pangenome controls, we efficiently filtered >97% of common SVs exclusive to 87 offspring. We find no evidence of increased autosomal SV burden for probands when compared to unaffected siblings yet observe a suggestive trend toward an increased SV burden on the X chromosome among affected females. We establish a workflow to prioritize potential pathogenic variants by integrating autism risk genes and putative noncoding regulatory elements defined from ATAC-seq and CUT&Tag data from the developing cortex. In total, we identified three pathogenic variants in TBL1XR1 , MECP2 , and SYNGAP1 , as well as nine candidate de novo and biallelic inherited homozygous SVs, most of which were missed by short-read sequencing. Our work highlights the potential of phased genomes to discover complex more pathogenic mutations and the power of the pangenome to restrict the focus on an increasingly smaller number of SVs for clinical evaluation.

Oncogenesis involves widespread genetic and epigenetic alterations, yet the full spectrum of somatic variation genome-wide remains unresolved. We generated a near-telomere-to-telomere (T2T) diploid assembly of a donor paired with deep short- and long-read sequencing of their melanoma. This revealed that 16% of somatic variants occur in sequences absent from GRCh38, with satellite repeats acting as hotspots for UV-induced damage due to sequence-intrinsic mutability and inefficient repair. Centromere kinetochore domains emerged as focal sites of structural, genetic, and epigenetic variation, leading to remodeling of centromere kinetochore binding domains during tumor evolution. Single-molecule telomere reconstructions uncovered cycles of attrition, deletion, and telomerase-mediated extension that shape cancer telomeres. Finally, diploid chromatin maps exposed that copy number alterations and epimutations, rather than point mutations, predominate in rewiring cancer regulatory programs. These findings define the full landscape of a cancer’s somatic variation and their functional impact, establishing a blueprint for T2T studies of mosaicism.

The All of Us Research Program (AoU) is a national biobank seeking to enroll one million individuals in the United States to link genomic and biomedical data, including short- and long-read whole-genome sequencing (srWGS/LRS), with rich electronic health record (EHR) information. Here, we present the first large-scale analyses of long-read sequencing (LRS) in AoU and offer a new framework for deriving genomic insights into complex structural variation (SV) of relevance to human health and disease. We performed joint analyses of 1,027 individuals self-identifying as Black or African American, sequenced to ~8x coverage with Pacific Biosciences HiFi technology and processed using cloud-native pipelines. From these LRS data we constructed a comprehensive variant callset encompassing known (FMR1 and HTT) and novel repeat expansions, clinically relevant haplotypes at loci inaccessible to srWGS, and haplotypes relevant to disease risk (HLA) and pharmacogenomics (CYP2D6), including SNVs, indels, and SVs. We developed methods for cohort-level variant calling and a scalable workflow to impute >750,000 of these SVs into existing srWGS datasets for trait association and human disease studies. Expanding to 10,000 self-identified Black or African American AoU participants with srWGS and matched EHRs, we identified 291 SV-disease associations (p < 1e-5) spanning 226 conditions with 50.9% of associations involving SVs absent from the matched srWGS callset. Across the 226 traits, after fine-mapping using SVs and SNVs we identified 191 SV-disease pairs spanning 160 traits (70.8%) where the SV had the strongest association within the locus. Associations specific to those with computed ancestry similar to the African reference population exhibited larger effect sizes and lower allele frequencies, consistent with high-risk, ancestry-specific variants. These results demonstrate that the integration of LRS into AoU and future biobank initiatives can provide transformative new insights into genomic variation with potentially profound impact on precision medicine.

The most common genomic disorder, chromosome 22q11.2 microdeletion syndrome (22q11.2DS), is mediated by highly identical and polymorphic segmental duplications (SDs) known as low copy repeats (LCRs; regions A-D) that have been challenging to sequence and characterize. Here, we report the sequence-resolved genomic architecture of 135 chromosome 22q11.2 haplotypes from diverse 1000 Genomes Project samples. We find that more than 90% of the copy number variation is polarized to the most proximal LCR region A (LCRA) where 50 distinct structural configurations are observed (∼189 kbp to ∼2.15 Mbp or 11-fold length variation). A higher-order SD cassette structure of 105 kbp in length, flanked by 25 kbp long inverted repeats, drives this variation and emerged in the human–chimpanzee ancestral lineage later expanding in humans ∼1.0 [0.8-1.2] million years ago. African LCRA haplotypes are significantly longer (p=0.0047) when compared to non-Africans yet are predicted to be more protected against recurrent microdeletions (p=0.00053) due to a preponderance of flanking SDs in an inverted orientation. Conversely, we identified nine distinct inversion polymorphisms, including five recurrent ∼2.28 Mbp inversions extending across the critical region (LCRA-D) and four smaller inversions (two LCRA-B, one LCRC-D, and one LCRB-D); 7/9 of these events were identified in haplotypes of African and admixed American ancestry. Finally, we sequence and assemble four families and show that LCRA-D deletion breakpoints map to the 105 kbp repeat unit while inversion breakpoints associate with the 25 kbp repeats adjacent to palindromic AT-rich regions. In one family, we observe evidence of more complex unequal crossover events associated with gene conversion and multiple breakpoints. Our findings suggest that specific haplotype configurations are protective and susceptible to chromosome 22q11.2DS while recurrent large-scale inversions help to explain why this syndrome is less prevalent among individuals of African descent.

The NPIP (nuclear pore interacting protein) gene family has expanded to high copy number in humans and African apes where it has been subject to an excess of amino acid replacement consistent with positive selection (1). Due to the limitations of short-read sequencing, NPIP human genetic diversity has been poorly understood. Using highly accurate assemblies generated from long-read sequencing as part of the human pangenome, we completely characterize 169 human haplotypes (4,665 NPIP paralogs and alleles). Of the 28 NPIP paralogs, just three ( NPIPB2 , B11 , and B14 ) are fixed at a single copy, and only a single locus, B2 , shows no structural variation. Four NPIP paralogs map to large segmental duplication blocks that mediate polymorphic inversions (355 kbp–1.6 Mbp) corresponding to microdeletions associated with developmental delay and autism. Haplotype-based tests of positive selection and selective sweeps identify two paralogs, B9 and B15 , within the top percentile for both tests. Using full-length cDNA data from 101 tissue/cell types, we construct paralog-specific gene models and show that 56% (31/55 most abundant isoforms) have not been previously described in RefSeq. We define six distinct translation start sites and other protein structural features that distinguish paralogs, including a variable number tandem repeat that encodes a beta helix of variable size that emerged ∼3.1 million years ago in human evolution. Among the 28 NPIP paralogs, we identify distinct tissue and developmental patterns of expression with only a few maintaining the ancestral testis-enriched expression. A subset of paralogs ( NPIPA1 , A5 , A6-9 , B3-5 , and B12/B13 ) show increased brain expression. Our results suggest ongoing positive selection in the human population and rapid diversification of NPIP gene models.

Motivation: Centromeres are chromosomal regions historically understudied with sequencing technologies due to their repetitive nature and short-read mapping limitations. However, recent improvements in long-read sequencing allow for the investigation of complex regions of the genome at the sequence and epigenetic levels. Results: Here, we present Centromere Dip Region (CDR)-Finder: a tool to identify regions of hypomethylation within the centromeres of high-quality, contiguous genome assemblies. These regions are typically associated with a unique type of chromatin containing the histone H3 variant CENP-A, which marks the location of the kinetochore. CDR-Finder identifies the CDRs in large and short centromeres and generates a BED file indicating the location of the CDRs within the centromere. It also outputs a plot for visualization, validation, and downstream analysis. Availability and implementation: CDR-Finder is available at https://github.com/EichlerLab/CDR-Finder.

Centromeres are chromosomal regions historically understudied with sequencing technologies due to their repetitive nature and short-read mapping limitations. However, recent improvements in long-read sequencing allowed for the investigation of complex regions of the genome at the sequence and epigenetic levels. Here, we present Centromere Dip Region (CDR)-Finder: a tool to identify regions of hypomethylation within the centromeres of high-quality, contiguous genome assemblies. These regions are typically associated with a unique type of chromatin containing the histone H3 variant CENP-A, which marks the location of the kinetochore. CDR-Finder identifies the CDRs in large and short centromeres and generates a BED file indicating the location of the CDRs within the centromere. It also outputs a plot for visualization, validation, and downstream analysis. CDR-Finder is available at https://github.com/EichlerLab/CDR-Finder.

Haplotype information is crucial for biomedical and population genetics research. However, current strategies to produce de novo haplotype-resolved assemblies often require either difficult-to-acquire parental data or an intermediate haplotype-collapsed assembly. Here, we present Graphasing, a workflow which synthesizes the global phase signal of Strand-seq with assembly graph topology to produce chromosome-scale de novo haplotypes for diploid genomes. Graphasing readily integrates with any assembly workflow that both outputs an assembly graph and has a haplotype assembly mode. Graphasing performs comparably to trio phasing in contiguity, phasing accuracy, and assembly quality, outperforms Hi-C in phasing accuracy, and generates human assemblies with over 18 chromosome-spanning haplotypes.