Philip C. Dishuck

Protein-coding genes mapping to high-identity segmental duplications (SDs) have been difficult to annotate and characterize and are the source of most previously unknown protein-coding genes being discovered as part of the human pangenome. Here, we combine long-read assembled human genomes (298) and long-read transcriptome data (5.6 billion full-length cDNA from 83 tissues) to phylogenetically interrogate 493 gene families discovering 2713 potentially copy number polymorphic genes not present in the human reference genome. For reference SD gene families where paralog specificity can be assigned, we find that 60.0% are expressed and maintain open reading frames, with 45.7% showing high expression in brain, embryo, or testis. We revise 386 gene models, including 150 that absent or different from current T2T-CHM13 gene annotation and 236 (35.1%) pseudogenes as protein-coding where we find evidence of transcription, an open reading frame, and chromatin-accessible promoters. We find that 24.2% of SD genes show evidence of constraint for both copy number and amino acid mutation. The majority of these constraint genes are ancestral, whereas only 16.2% of derived duplicated genes that emerged recently in the human lineage show evidence of constraint. The pangenome provides unparalleled specificity to understand genetic variation in SD genes allowing us to distinguish functional genes from pseudogenes and highlighting potential gene innovations that arose most recently in human 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.

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.

Motivation: We are now in the era of being able to routinely generate highly contiguous (near telomere-to-telomere) genome assemblies of human and nonhuman species. Complex structural variation and regions of rapid evolutionary turnover are being discovered for the first time. Thus, efficient and informative visualization tools are needed to evaluate and directly observe structural differences between two or more genomes. Results: We developed SVbyEye, an open-source R package to visualize and annotate sequence-to-sequence alignments along with various functionalities to process these alignments. The tool facilitates the characterization of complex structural variants in the context of sequence homology helping resolve the mechanisms underlying their formation. Availability and implementation: SVbyEye is available on GitHub (https://github.com/daewoooo/SVbyEye) and via Zenodo (https://doi.org/10.5281/zenodo.15303553).

SUMMARY: NOTCH2NL ( NOTCH2 -N-terminus-like) genes arose from ape-specific chromosome 1 segmental duplications implicated in human brain cortical expansion, including an incomplete NOTCH2 gene. Genetic characterization of these loci and their regulation is complicated because they are embedded in large, nearly identical duplications that predispose to recurrent microdeletion syndromes. Using near-complete long-read assemblies generated from 70 human and 12 ape haploid genomes, we show independent recurrent duplication among apes with protein-coding copies emerging in humans 2.2-3.7 million years ago. We distinguish NOTCH2NL paralogs present in every human haplotype ( NOTCH2NLA ) from copy number variable ones. We also characterize large-scale structural variation, including gene conversion, for 28% of haplotypes leading to a previously undescribed paralog, NOTCH2tv. Finally, we apply Fiber-seq and long-read transcript sequencing to human dorsal forebrain organoids to characterize the regulatory landscape and find that the most fixed paralogs, NOTCH2 and NOTCH2NLA , harbor the greatest number of paralog-specific elements potentially driving their regulation.

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 We are now in the era of being able to routinely generate highly contiguous (near telomere-to-telomere) genome assemblies of human and nonhuman species. Complex structural variation and regions of rapid evolutionary turnover are being discovered for the first time. Thus, efficient and informative visualization tools are needed to evaluate and directly observe structural differences between two or more genomes. Results We developed SVbyEye, an open-source R package to visualize and annotate sequence-to-sequence alignments along with various functionalities to process alignments in PAF format. The tool facilitates the characterization of complex structural variants in the context of sequence homology helping resolve the mechanisms underlying their formation. Availability and implementation SVbyEye is available at https://github.com/daewoooo/SVbyEye.

TBC1D3is a primate-specific gene family that has expanded in the human lineage and has been implicated in neuronal progenitor proliferation and expansion of the frontal cortex. The gene family and its expression have been challenging to investigate because it is embedded in high-identity and highly variable segmental duplications. We sequenced and assembled the gene family using long-read sequencing data from 34 humans and 11 nonhuman primate species. Our analysis shows that this particular gene family has independently duplicated in at least five primate lineages, and the duplicated loci are enriched at sites of large-scale chromosomal rearrangements on Chromosome 17. We find that all human copy-number variation maps to two distinct clusters located at Chromosome 17q12 and that humans are highly structurally variable at this locus, differing by as many as 20 copies and ∼1 Mbp in length depending on haplotypes. We also show evidence of positive selection, as well as a significant change in the predicted human TBC1D3 protein sequence. Last, we find that, despite multiple duplications, humanTBC1D3expression is limited to a subset of copies and, most notably, from a single paralog group:TBC1D3-CDKL. These observations may help explain why a gene potentially important in cortical development can be so variable in the human population.

TBC1D3 is a primate-specific gene family that has expanded in the human lineage and has been implicated in neuronal progenitor proliferation and expansion of the frontal cortex. The gene family and its expression have been challenging to investigate because it is embedded in high-identity and highly variable segmental duplications. We sequenced and assembled the gene family using long-read sequencing data from 34 humans and 11 nonhuman primate species. Our analysis shows that this particular gene family has independently duplicated in at least five primate lineages, and the duplicated loci are enriched at sites of large-scale chromosomal rearrangements on chromosome 17. We find that most humans vary along two TBC1D3 clusters where human haplotypes are highly variable in copy number, differing by as many as 20 copies, and structure (structural heterozygosity 90%). We also show evidence of positive selection, as well as a significant change in the predicted human TBC1D3 protein sequence. Lastly, we find that, despite multiple duplications, human TBC1D3 expression is limited to a subset of copies and, most notably, from a single paralog group: TBC1D3-CDKL . These observations may help explain why a gene potentially important in cortical development can be so variable in the human population.

The prevalence of highly repetitive sequences within the human Y chromosome has prevented its complete assembly to date¹ and led to its systematic omission from genomic analyses. Here we present de novo assemblies of 43 Y chromosomes spanning 182,900 years of human evolution and report considerable diversity in size and structure. Half of the male-specific euchromatic region is subject to large inversions with a greater than twofold higher recurrence rate compared with all other chromosomes². Ampliconic sequences associated with these inversions show differing mutation rates that are sequence context dependent, and some ampliconic genes exhibit evidence for concerted evolution with the acquisition and purging of lineage-specific pseudogenes. The largest heterochromatic region in the human genome, Yq12, is composed of alternating repeat arrays that show extensive variation in the number, size and distribution, but retain a 1:1 copy-number ratio. Finally, our data suggest that the boundary between the recombining pseudoautosomal region 1 and the non-recombining portions of the X and Y chromosomes lies 500 kb away from the currently established¹ boundary. The availability of fully sequence-resolved Y chromosomes from multiple individuals provides a unique opportunity for identifying new associations of traits with specific Y-chromosomal variants and garnering insights into the evolution and function of complex regions of the human genome.