Biology subjects

Munson, K. M.

Publications and source records attributed to Munson, K. M..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

A near telomere-to-telomere phased reference assembly for the male mountain gorilla

The endangered mountain gorilla, Gorilla beringei beringei, faces numerous threats to its survival, highlighting the urgent need for genomic resources to aid conservation efforts. Here, we present a near telomere-to-telomere, haplotype-phased reference genome assembly for a male mountain gorilla generated using PacBio HiFi (26.77x ave. coverage) and Oxford Nanopore Technologies (52.87x ave. coverage) data. The resulting non-scaffolded assembly exhibits exceptional contiguity, with contig N50 of [~]95 Mbp for the combined pseudohaplotype (3,540,458,497 bp), 56.5 Mbp (3.1 Gbp) and 51.0 Mbp (3.2 Gbp) for each haplotype, an average QV of 65.15 (error rate = 3.1 x 10-7), and a BUSCO score of 98.4%. These represent substantial improvements over most other available primate genomes. This first high-quality reference genome of the mountain gorilla provides an invaluable resource for future studies on gorilla evolution, adaptation, and conservation, ultimately contributing to the long-term survival of this iconic species.

genomics↗

bioRxiv2024

The Platinum Pedigree: A long-read benchmark for genetic variants

Recent advances in genome sequencing have improved variant calling in complex regions of the human genome. However, it is difficult to quantify variant calling performance since existing standards often focus on specificity, neglecting completeness in difficult to analyze regions. To create a more comprehensive truth set, we used Mendelian inheritance in a large pedigree (CEPH-1463) to filter variants across Illumina, PacBio high-fidelity (HiFi), and Oxford Nanopore Technologies platforms. This generated a variant map with over 4.7 million single-nucleotide variants, 767,795 indels, 537,486 tandem repeats, and 24,315 structural variants, covering 2.77 Gb of the GRCh38 genome. This work adds [~]200 Mb of high-confidence regions, including 8% more small variants, and introduces the first tandem repeat and structural variant truth sets for NA12878. As an example of the value of this improved benchmark, we retrained DeepVariant using this data to reduce genotyping errors by [~]34%.

genomics↗

bioRxiv2024

Complex genetic variation in nearly complete human genomes

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 gaps1,2 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 -satellite high-order repeat (HOR) array length and characterize the pattern of mobile element insertions into -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 reference1 significantly enhances genotyping accuracy from short-read data, enabling whole-genome inference3 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.

genomics↗

bioRxiv2024

A familial, telomere-to-telomere reference for human de novo mutation and recombination from a four-generation pedigree

Using five complementary short- and long-read sequencing technologies, we phased and assembled >95% of each diploid human genome in a four-generation, 28-member family (CEPH 1463) allowing us to systematically assess de novo mutations (DNMs) and recombination. From this family, we estimate an average of 192 DNMs per generation, including 75.5 de novo single-nucleotide variants (SNVs), 7.4 non-tandem repeat indels, 79.6 de novo indels or structural variants (SVs) originating from tandem repeats, 7.7 centromeric de novo SVs and SNVs, and 12.4 de novo Y chromosome events per generation. STRs and VNTRs are the most mutable with 32 loci exhibiting recurrent mutation through the generations. We accurately assemble 288 centromeres and six Y chromosomes across the generations, documenting de novo SVs, and demonstrate that the DNM rate varies by an order of magnitude depending on repeat content, length, and sequence identity. We show a strong paternal bias (75-81%) for all forms of germline DNM, yet we estimate that 17% of de novo SNVs are postzygotic in origin with no paternal bias. We place all this variation in the context of a high-resolution recombination map ([~]3.5 kbp breakpoint resolution). We observe a strong maternal recombination bias (1.36 maternal:paternal ratio) with a consistent reduction in the number of crossovers with increasing paternal (r=0.85) and maternal (r=0.65) age. However, we observe no correlation between meiotic crossover locations and de novo SVs, arguing against non-allelic homologous recombination as a predominant mechanism. The use of multiple orthogonal technologies, near-telomere-to-telomere phased genome assemblies, and a multi-generation family to assess transmission has created the most comprehensive, publicly available "truth set" of all classes of genomic variants. The resource can be used to test and benchmark new algorithms and technologies to understand the most fundamental processes underlying human genetic variation.

genomics↗

bioRxiv2024

Resolving the chromatin impact of mosaic variants with targeted Fiber-seq

Accurately quantifying the functional consequences of non-coding mosaic variants requires the pairing of DNA sequence with both accessible and closed chromatin architectures along individual DNA molecules--a pairing that cannot be achieved using traditional fragmentation-based chromatin assays. We demonstrate that targeted single-molecule chromatin fiber sequencing (Fiber-seq) achieves this, permitting single-molecule, long-read genomic and epigenomic profiling across targeted >100 kilobase loci with [~]10-fold enrichment over untargeted sequencing. Targeted Fiber-seq reveals that pathogenic expansions of the DMPK CTG repeat that underlie Myotonic Dystrophy 1 are characterized by somatic instability and disruption of multiple nearby regulatory elements, both of which are repeat length-dependent. Furthermore, we reveal that therapeutic adenine base editing of the segmentally duplicated {gamma}-globin (HBG1/HBG2) promoters in primary human hematopoietic cells induced towards an erythroblast lineage increases the accessibility of the HBG1 promoter as well as neighboring regulatory elements. Overall, we find that these non-protein coding mosaic variants can have complex impacts on chromatin architectures, including extending beyond the regulatory element harboring the variant.

genomics↗

bioRxiv2024

A haplotype-resolved view of human gene regulation

Diploid human cells contain two non-identical genomes, and differences in their regulation underlie human development and disease. We present Fiber-seq Inferred Regulatory Elements (FIRE) and show that FIRE provides a more comprehensive and quantitative snapshot of the accessible chromatin landscape across the 6 Gbp diploid human genome, overcoming previously unrecognized biases in existing regulatory element catalogs. FIRE enables comprehensive detection of haplotype-selective chromatin accessibility (HSCA), exposing novel imprinted elements lacking underlying parent-of-origin CpG methylation differences, and gene regulatory modules that permit genes to escape X chromosome inactivation. We uncover that the human leukocyte antigen (HLA) locus harbors the most HSCA in immune cells, where we resolve specific transcription factor (TF) binding events disrupted by disease-associated variants. Finally, we demonstrate that the regulatory landscape of a cell is littered with autosomal somatic chromatin epimutations that are propagated by clonal expansions to create mitotically stable and non-genetically deterministic chromatin alterations.

genomics↗

bioRxiv2024

Structural polymorphism and diversity of human segmental duplications

Segmental duplications (SDs) contribute significantly to human disease, evolution, and diversity yet have been difficult to resolve at the sequence level. We present a population genetics survey of SDs by analyzing 170 human genome assemblies where the majority of SDs are fully resolved using long-read sequence assembly. Excluding the acrocentric short arms, we identify 173.2 Mbp of duplicated sequence (47.4 Mbp not present in the telomere-to-telomere reference) distinguishing fixed from structurally polymorphic events. We find that intrachromosomal SDs are among the most variable with rare events mapping near their progenitor sequences. African genomes harbor significantly more intrachromosomal SDs and are more likely to have recently duplicated gene families with higher copy number when compared to non-African samples. A comparison to a resource of 563 million full-length Iso-Seq reads identifies 201 novel, potentially protein-coding genes corresponding to these copy number polymorphic SDs.

genomics↗

bioRxiv2024

Structural and genetic diversity in the secreted mucins, MUC5AC and MUC5B

The secreted mucins MUC5AC and MUC5B play critical defensive roles in airway pathogen entrapment and mucociliary clearance by encoding large glycoproteins with variable number tandem repeats (VNTRs). These polymorphic and degenerate protein coding VNTRs make the loci difficult to investigate with short reads. We characterize the structural diversity of MUC5AC and MUC5B by long-read sequencing and assembly of 206 human and 20 nonhuman primate (NHP) haplotypes. We find that human MUC5B is largely invariant (5761-5762aa); however, seven haplotypes have expanded VNTRs (6291-7019aa). In contrast, 30 allelic variants of MUC5AC encode 16 distinct proteins (5249-6325aa) with cysteine-rich domain and VNTR copy number variation. We grouped MUC5AC alleles into three phylogenetic clades: H1 (46%, [~]5654aa), H2 (33%, [~]5742aa), and H3 (7%, [~]6325aa). The two most common human MUC5AC variants are smaller than NHP gene models, suggesting a reduction in protein length during recent human evolution. Linkage disequilibrium (LD) and Tajimas D analyses reveal that East Asians carry exceptionally large MUC5AC LD blocks with an excess of rare variation (p<0.05). To validate this result, we used Locityper for genotyping MUC5AC haplogroups in 2,600 unrelated samples from the 1000 Genomes Project. We observed signatures of positive selection in H1 and H2 among East Asians and a depletion of the likely ancestral haplogroup (H3). In Africans and Europeans, H3 alleles show an excess of common variation and deviate from Hardy-Weinberg equilibrium, consistent with heterozygote advantage and balancing selection. This study provides a generalizable strategy to characterize complex protein coding VNTRs for improved disease associations.

genomics↗

bioRxiv2024

Independent expansion, selection and hypervariability of the TBC1D3 gene family in humans

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.

genetics↗

bioRxiv2024

Complete chromosome 21 centromere sequences from a Down syndrome family reveal size asymmetry and differences in kinetochore attachment

Down syndrome, the most common form of human intellectual disability, is caused by nondisjunction and chromosome 21 trisomy (T21). Small centromeres have been hypothesized to contribute to its aetiology and studies on mammals suggest that larger centromeres are more efficiently transmitted, yet complete sequencing of chromosome 21 (chr21) centromeres has been particularly challenging. Using long-read sequencing, we sequenced and assembled the centromeres from eight families that include a child with free T21 (1 trio, 6 child-mother duos, and 1 singleton) all resulting from maternal meiosis I errors. Two of these families carry the smallest chr21 centromeres (143 and 181 kbp) observed in female individuals to date, exhibiting a [~]10.7- and [~]19.4-fold centromeric -satellite higher-order repeat array size difference between the maternally inherited homologs, respectively. In both cases, the longer centromere harbors a poorly defined centromere dip region, marked by DNA hypomethylation, in the proband but not in the mother. A comparison of all proband chr21 centromeres (n=24) to those of controls (n=261) shows that small centromeres are not enriched in families with T21 (p-value=0.73); contrarily, chr21 extreme centromere size asymmetry (>10-fold) is unique of T21 (p-value=0.003), suggesting that this feature may represent a genetic risk factor for a subset of families with free T21. Additionally, phylogenetic reconstruction reveals that human chr21 has been particularly prone to such variation with some of the biggest size differences occurring over the last [~]17 thousand years of human evolution.

genomics↗

bioRxiv2023

Synchronized long-read genome, methylome, epigenome, and transcriptome for resolving a Mendelian condition

Resolving the molecular basis of a Mendelian condition (MC) remains challenging owing to the diverse mechanisms by which genetic variants cause disease. To address this, we developed a synchronized long-read genome, methylome, epigenome, and transcriptome sequencing approach, which enables accurate single-nucleotide, insertion-deletion, and structural variant calling and diploid de novo genome assembly, and permits the simultaneous elucidation of haplotype-resolved CpG methylation, chromatin accessibility, and full-length transcript information in a single long-read sequencing run. Application of this approach to an Undiagnosed Diseases Network (UDN) participant with a chromosome X;13 balanced translocation of uncertain significance revealed that this translocation disrupted the functioning of four separate genes (NBEA, PDK3, MAB21L1, and RB1) previously associated with single-gene MCs. Notably, the function of each gene was disrupted via a distinct mechanism that required integration of the four omes to resolve. These included nonsense-mediated decay, fusion transcript formation, enhancer adoption, transcriptional readthrough silencing, and inappropriate X chromosome inactivation of autosomal genes. Overall, this highlights the utility of synchronized long-read multi-omic profiling for mechanistically resolving complex phenotypes.

genetics↗

bioRxiv2023

The variation and evolution of complete human centromeres

We completely sequenced and assembled all centromeres from a second human genome and used two reference sets to benchmark genetic, epigenetic, and evolutionary variation within centromeres from a diversity panel of humans and apes. We find that centromere single-nucleotide variation can increase by up to 4.1-fold relative to other genomic regions, with the caveat that up to 45.8% of centromeric sequence, on average, cannot be reliably aligned with current methods due to the emergence of new -satellite higher-order repeat (HOR) structures and two to threefold differences in the length of the centromeres. The extent to which this occurs differs depending on the chromosome and haplotype. Comparing the two sets of complete human centromeres, we find that eight harbor distinctly different -satellite HOR array structures and four contain novel -satellite HOR variants in high abundance. DNA methylation and CENP-A chromatin immunoprecipitation experiments show that 26% of the centromeres differ in their kinetochore position by at least 500 kbp--a property not readily associated with novel -satellite HORs. To understand evolutionary change, we selected six chromosomes and sequenced and assembled 31 orthologous centromeres from the common chimpanzee, orangutan, and macaque genomes. Comparative analyses reveal nearly complete turnover of -satellite HORs, but with idiosyncratic changes in structure characteristic to each species. Phylogenetic reconstruction of human haplotypes supports limited to no recombination between the p- and q-arms of human chromosomes and reveals that novel -satellite HORs share a monophyletic origin, providing a strategy to estimate the rate of saltatory amplification and mutation of human centromeric DNA.

genomics↗

bioRxiv2023

Whole-genome long-read sequencing downsampling and its effect on variant calling precision and recall

Advances in long-read sequencing (LRS) technology continue to make whole-genome sequencing more complete, affordable, and accurate. LRS provides significant advantages over short-read sequencing approaches, including phased de novo genome assembly, access to previously excluded genomic regions, and discovery of more complex structural variants (SVs) associated with disease. Limitations remain with respect to cost, scalability, and platform-dependent read accuracy and the tradeoffs between sequence coverage and sensitivity of variant discovery are important experimental considerations for the application of LRS. We compare the genetic variant calling precision and recall of Oxford Nanopore Technologies (ONT) and PacBio HiFi platforms over a range of sequence coverages. For read-based applications, LRS sensitivity begins to plateau around 12-fold coverage with a majority of variants called with reasonable accuracy (F1 score above 0.5), and both platforms perform well for SV detection. Genome assembly increases variant calling precision and recall of SVs and indels in HiFi datasets with HiFi outperforming ONT in quality as measured by the F1 score of assembly-based variant callsets. While both technologies continue to evolve, our work offers guidance to design cost-effective experimental strategies that do not compromise on discovering novel biology.

genomics↗

bioRxiv2023

Conservation of chromatin organization within human and primate centromeres

The focal attachment of the kinetochore to the centromere is essential for genome maintenance, yet the highly repetitive nature of satellite regional centromeres, such as those in humans, limits our understanding of their chromatin organization. We demonstrate that single-molecule chromatin fiber sequencing (Fiber-seq) can uniquely co-resolve kinetochore and surrounding chromatin architectures along point centromeres, revealing largely homogeneous single-molecule kinetochore occupancy along each chromosome. In contrast, extension of Fiber-seq to regional satellite centromeres exposed marked per-molecule heterogeneity in their chromatin organization. Regional CENP-A-marked centromere cores uniquely contain a dichotomous chromatin organization (dichromatin) composed of compacted nucleosome arrays punctuated with highly accessible chromatin patches. CENP-B occupancy phases dichromatin to the underlying alpha-satellite repeat within centromere cores, but is not necessary for dichromatin formation. Centromere core dichromatin is a conserved feature between humans despite the marked divergence of their underlying alpha-satellite organization and is similarly a conserved feature along regional centromeres that lack satellite repeats in gibbon. Overall, the chromatin organization of regional centromeres is defined by marked per-molecule heterogeneity, likely buffering kinetochore attachment against sequence and structural variability within regional centromeres. HighlightsO_LIDichotomous accessible and compacted chromatin (dichromatin) marks centromere cores C_LIO_LIHighly accessible chromatin patches punctuate sites of kinetochore attachment C_LIO_LIDichromatin can form irrespective of CENP-B occupancy C_LIO_LIConservation within centromeres is mediated at the level of chromatin, not DNA C_LI

genomics↗

bioRxiv2023

Structurally divergent and recurrently mutated regions of primate genomes

To better understand the pattern of primate genome structural variation, we sequenced and assembled using multiple long-read sequencing technologies the genomes of eight nonhuman primate species, including New World monkeys (owl monkey and marmoset), Old World monkey (macaque), Asian apes (orangutan and gibbon), and African ape lineages (gorilla, bonobo, and chimpanzee). Compared to the human genome, we identified 1,338,997 lineage-specific fixed structural variants (SVs) disrupting 1,561 protein-coding genes and 136,932 regulatory elements, including the most complete set of human-specific fixed differences. Across 50 million years of primate evolution, we estimate that 819.47 Mbp or ~27% of the genome has been affected by SVs based on analysis of these primate lineages. We identify 1,607 structurally divergent regions (SDRs) wherein recurrent structural variation contributes to creating SV hotspots where genes are recurrently lost (CARDs, ABCD7, OLAH) and new lineage-specific genes are generated (e.g., CKAP2, NEK5) and have become targets of rapid chromosomal diversification and positive selection (e.g., RGPDs). High-fidelity long-read sequencing has made these dynamic regions of the genome accessible for sequence-level analyses within and between primate species for the first time.

genomics↗

bioRxiv2022

Assembly of 43 diverse human Y chromosomes reveals extensive complexity and variation

The prevalence of highly repetitive sequences within the human Y chromosome has led to its incomplete assembly and systematic omission from genomic analyses. Here, we present long-read de novo assemblies of 43 diverse Y chromosomes spanning 180,000 years of human evolution, including two from deep-rooted African Y lineages, and report remarkable complexity and diversity in chromosome size and structure, in contrast with its low level of base substitution variation. The size of the Y chromosome assemblies varies extensively from 45.2 to 84.9 Mbp and include, on average, 81 kbp of novel sequence per Y chromosome. Half of the male-specific euchromatic region is subject to large inversions with a >2-fold higher recurrence rate compared to inversions in the rest of the human genome. Ampliconic sequences associated with these inversions further show differing mutation rates that are sequence context-dependent and some ampliconic genes show evidence for concerted evolution with the acquisition and purging of lineage-specific pseudogenes. The largest heterochromatic region in the human genome, the Yq12, is composed of alternating arrays of DYZ1 and DYZ2 repeat units that show extensive variation in the number, size and distribution of these arrays, but retain a 1:1 copy number ratio of the monomer repeats, consistent with the notion that functional or evolutionary forces are acting on this chromosomal region. Finally, our data suggests that the boundary between the recombining pseudoautosomal region 1 and the non-recombining portions of the X and Y chromosomes lies 500 kbp distal to the currently established boundary. The availability of sequence-resolved Y chromosomes from multiple individuals provides a unique opportunity for identifying new associations of specific traits with Y-chromosomal variants and garnering novel insights into the evolution and function of complex regions of the human genome.

genomics↗

bioRxiv2022

A Draft Human Pangenome Reference

The Human Pangenome Reference Consortium (HPRC) presents a first draft human pangenome reference. The pangenome contains 47 phased, diploid assemblies from a cohort of genetically diverse individuals. These assemblies cover more than 99% of the expected sequence and are more than 99% accurate at the structural and base-pair levels. Based on alignments of the assemblies, we generated a draft pangenome that captures known variants and haplotypes, reveals novel alleles at structurally complex loci, and adds 119 million base pairs of euchromatic polymorphic sequence and 1,529 gene duplications relative to the existing reference, GRCh38. Roughly 90 million of the additional base pairs derive from structural variation. Using our draft pangenome to analyze short-read data reduces errors when discovering small variants by 34% and boosts the detected structural variants per haplotype by 104% compared to GRCh38-based workflows, and by 34% compared to using previous diversity sets of genome assemblies.

genomics↗

bioRxiv2022

Increased mutation rate and interlocus gene conversion within human segmental duplications.

Single-nucleotide variants (SNVs) within segmental duplications (SDs) have not been systematically assessed because of the difficulty in mapping short-read sequence data to virtually identical repetitive sequences. Using 102 phased human haplotypes, we constructed 1:1 unambiguous alignments spanning high-identity SDs and compared the pattern of SNVs between unique and SD regions. We find that human SNVs are elevated 60% in SDs compared to unique regions. We estimate that at least 23% of this increase is due to interlocus gene conversion (IGC) with >7 Mbp 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 ~800 protein-coding genes. The latter includes 171 genes that have "relocated" on average 1.61 Mbp in a subset of human haplotypes. Using a coalescent framework, we show that SD regions are evolutionarily older when compared to unique sequences with most of this signal originating from putative IGC loci. SNVs within SDs, however, also exhibit a distinct mutational spectrum where there is a 27.1% increase in transversions that convert cytosine to guanine or the reverse across all triplet contexts. In addition, we observe a 7.6% reduction in the frequency of CpG associated mutations when compared to unique DNA. We hypothesize that these distinct mutational properties help to maintain an overall higher GC content of SD DNA when compared to unique DNA, and we show that these GC-favoring mutational events are likely driven by GC-biased conversion between paralogous sequences.

genomics↗