Biology subjects

Hartley, G. A.

Publications and source records attributed to Hartley, G. A..

13 recordsLinked to original sources

bioRxiv2026

Optimization of High Molecular Weight DNA Extractions from Dried, Museum-Grade Insects Enables Long-Read Sequencing, Phylogenetics, and Methylation Profiling

Developing an effective DNA extraction method that meets requirements for long-read sequencing of poorly preserved samples, such as museum specimens or ancient material, offers new opportunities for genomic analysis of endangered or extinct species for which samples are rare. However, these samples often yield degraded and highly fragmented DNA, rendering long-read sequencing infeasible for many specimens residing in museum collections. Herein, we demonstrate a protocol for successfully extracting DNA of sufficient quality for sequencing on the Oxford Nanopore Technologies long-read sequencing PromethION platform from a desiccated, museum-grade blue carpenter bee specimen (Xylocopa caerulea). We find the protocol is reproducible across specimens and yields high levels of long, endogenous X. caerulea-derived DNA, highlighting the utility of our method for enabling genomic studies of historical collections. From a single flow cell, we assembled the full-length mitochondrial genome and used this assembly to perform a phylogenetic analysis, accurately placing our X. caerulea specimen among related Xylocopa species, thus demonstrating the phylogenetic utility of long-read museomics. Using these long-read data, we analyzed native CpG methylation, finding endogenous methylation signals that correlate with genic and exonic sequences. This method expands the feasibility of genomic and epigenomic analyses from challenging samples, enhancing our ability to investigate the genomes of endangered and extinct species through archival resources.

genomics↗

bioRxiv2026

Genomes of two arid-zone marsupials uncover contrasting responses to climatic change

Small marsupials in the family Dasyuridae are a key component of Australias arid and semi-arid fauna, whose high species richness is proposed to reflect an opportunity-driven adaptive radiation. Despite growing interest in this group from both ecological and evolutionary perspectives, genomic data for most species is non-existent, or limited to a few marker loci. Here, we generated a chromosome-level reference genome and a de novo mitochondrial genome for the desert-dwelling Wongai ningaui (Ningaui ridei). The nuclear genome assembly is highly contiguous, with a scaffold N50 of 594.5 MB and high BUSCO gene recovery (93.8%). Additionally, we produced a draft assembly for the related, semi-arid slender-tailed dunnart (Sminthopsis murina). We then used these assemblies to explore the demographic histories of these species. We find evidence for contrasting patterns of population growth during the late Pleistocene and early Holocene, corresponding with differences in local climate, potentially consistent with differences in optimal habitat. The new genomic resources and demographic findings presented here provide a foundation for future studies on adaptive specialisation in this group of Australian marsupials. Significance StatementDasyurid marsupials are the primary carnivorous and insectivorous mammals in Australia. This family includes species such as the endangered Tasmanian devil (Sarcophilus harrisii) and quolls (Genus Dasyurus), as well as an emerging model species, the fat-tailed dunnart (Sminthopsis crassicaudata). Despite the species richness within dasyurids, most species remain under-studied. This is particularly true of arid and semi-arid zone species, who are often small in size, live in remote habitats and are cryptic by nature. By creating genome assemblies for two dasyurid species, this study provides resources to support phylogenetic, comparative and conservation research in arid zone marsupials. Importantly, the studys finding that arid and semi-arid species show distinct trajectories of demographic change in response to historical climate may have implications for the resilience of locally-adapted dasyurid species to ongoing climate change.

genomics↗

bioRxiv2025

Haplotype-Resolved Genomics Reveals Conserved Chromatin Architecture and Epigenetic Constraints of Human Neocentromeres

Human neocentromeres are functional centromeres demarcated by CENP-A nucleosomes that form ectopically at alpha satellite-free loci. How neocentromeres reshape local chromatin and which features of native centromeric chromatin are preserved are unknown. We generated gapless, haplotype-resolved assemblies of native and neocentromeres from three patient-derived cell lines. Integrating CpG methylation, CENP-A profiling, and single-molecule chromatin fiber sequencing, we reveal chromatin features that define the essential centromeric architecture reconstituted during neocentromere establishment. We find that a deletion within the satellite array encompassing the hypo-CpG methylation centromere dip regions (CDRs) led to native centromere inactivation, that neocentromeres harbor CDRs and a dichromatin architecture, recapitulating features of alpha-satellite centromeres, and that LINEs demarcate neocentromere boundaries, implicating transposable elements in restricting CENP-A domain spreading. Moreover, neocentromeric chromatin is incompatible with promoter-like chromatin states, redefining the regulatory landscape within genic regions. Finally, using haplotype-specific chromatin footprinting, we resolve CENP-A nucleosome chromatin architecture of active centromeres.

genomics↗

bioRxiv2025

A complete diploid human genome benchmark for personalized genomics

Human genome resequencing typically involves mapping reads to a reference genome to call variants; however, this approach suffers from both technical and reference biases, leaving many duplicated and structurally polymorphic regions of the genome unmapped. Consequently, existing variant benchmarks, generated by the same methods, fail to assess these complex regions. To address this limitation, we present a telomere-to-telomere genome benchmark that achieves near-perfect accuracy (i.e. no detectable errors) across 99.4% of the complete, diploid HG002 genome. This benchmark adds 701.4 Mb of autosomal sequence and both sex chromosomes (216.8 Mb), totaling 15.3% of the genome that was absent from prior benchmarks. We also provide a diploid annotation of genes, transposable elements, segmental duplications, and satellite repeats, including 39,144 protein-coding genes across both haplotypes. To facilitate application of the benchmark, we developed tools for measuring the accuracy of sequencing reads, phased variant call sets, and genome assemblies against a diploid reference. Genome-wide analyses show that state-of-the-art de novo assembly methods resolve 2-7% more sequence and outperform variant calling accuracy by an order of magnitude, yielding just one error per 100 kb across 99.9% of the benchmark regions. Adoption of genome-based benchmarking is expected to accelerate the development of cost-effective methods for complete genome sequencing, expanding the reach of genomic medicine to the entire genome and enabling a new era of personalized genomics.

genomics↗

bioRxiv2024

Centromeric transposable elements and epigenetic status drive karyotypic variation in the eastern hoolock gibbon

Great apes have maintained a stable karyotype with few large-scale rearrangements; in contrast, gibbons have undergone a high rate of chromosomal rearrangements coincident with rapid centromere turnover. Here we characterize assembled centromeres in the Eastern hoolock gibbon, Hoolock leuconedys (HLE), finding a diverse group of transposable elements (TEs) that differ from the canonical alpha satellites found across centromeres of other apes. We find that HLE centromeres contain a CpG methylation centromere dip region, providing evidence this epigenetic feature is conserved in the absence of satellite arrays; nevertheless, we report a variety of atypical centromeric features, including protein-coding genes and mismatched replication timing. Further, large structural variations define HLE centromeres and distinguish them from other gibbons. Combined with differentially methylated TEs, topologically associated domain boundaries, and segmental duplications at chromosomal breakpoints, we propose that a "perfect storm" of multiple genomic attributes with propensities for chromosome instability shaped gibbon centromere evolution.

genomics↗

bioRxiv2024

Exploring Alu-Driven DNA Transductions in the Primate Genomes

Long terminal repeats (LTRs) and non-LTRs retrotransposons, aka retroelements, collectively occupy a substantial part of the human genome. Certain non-LTR retroelements, such as L1 and SVA, have the potential for DNA transduction, which involves the concurrent mobilization of flanking non-transposon DNA during retrotransposition. These events can be detected by computational approaches. Despite being the most abundant short interspersed sequences (SINEs) that are still active within the genomes of humans and other primates, the transduction rate caused by Alu sequences remains unexplored. Therefore, we conducted an analysis to address this research gap and utilized an in-house program to probe for the presence of Alu-related transductions in the human genome. We analyzed 118,489 full-length AluY subfamilies annotated within the first complete human reference genome, T2T-CHM13. For comparative insights, we extended our exploration to two non-human primate genomes, the chimpanzee and the rhesus monkey. After manual curation, our findings did not confirm any Alu-mediated transductions, whose source genes are, unlike L1 or SVA, transcribed by RNA polymerase III, implying that they are infrequent or possibly absent not only in the human but also in chimpanzee and rhesus monkey genomes. Although we identified loci in which the 3 Target Site Duplication (TSD) was located distantly from the retrotransposed AluYs, a transduction hallmark, our study could not find further support for such events. The observation of these instances can be explained by the incorporation of other nucleotides into the poly(A) tails in conjunction with polymerase slippage.

genetics↗

bioRxiv2023

The Complete Sequence and Comparative Analysis of Ape Sex Chromosomes

Apes possess two sex chromosomes--the male-specific Y and the X shared by males and females. The Y chromosome is crucial for male reproduction, with deletions linked to infertility1. The X chromosome carries genes vital for reproduction and cognition2. Variation in mating patterns and brain function among great apes suggests corresponding differences in their sex chromosomes. However, due to their highly repetitive nature and incomplete reference assemblies, ape sex chromosomes have been challenging to study. Here, using the methodology developed for the telomere-to-telomere (T2T) human genome, we produced gapless assemblies of the X and Y chromosomes for five great apes (chimpanzee, bonobo, gorilla, Bornean and Sumatran orangutans) and a lesser ape, the siamang gibbon. These assemblies allowed us to untangle the intricacies of ape sex chromosome evolution. We found that, compared to the Xs, the ape Ys vary greatly in size and have low alignability and high levels of structural rearrangements. This divergence on the Y arises from the accumulation of lineage-specific ampliconic regions, palindromes, transposable elements, and satellites. Our analysis of Y chromosome genes revealed expansions of multi-copy gene families and signatures of purifying selection. Thus, the Y exhibits dynamic evolution, while the X is more stable. Mapping short-read sequencing data to these assemblies revealed diversity and selection patterns on sex chromosomes of >100 great ape individuals. These reference assemblies are expected to inform human evolution and conservation genetics of nonhuman apes, all of which are endangered species.

genomics↗

bioRxiv2023

Reference genome of the endangered eastern quoll (Dasyurus viverrinus)

The eastern quoll (Dasyurus viverrinus) is an endangered marsupial mesopredator native to Australia. Since the extirpation of the last mainland Australian populations in the late 20th century, wild populations of this species have been restricted to two islands at the far southern end of its historical range. Eastern quolls are the subject of captive breeding programs and attempts have recently been made to re-establish a population in mainland Australia through translocations. However, few resources currently exist to guide the genetic management of this species. Here, we generated a chromosome-scale reference genome for the eastern quoll, along with gene annotations supported by multi-tissue transcriptomes. Through comparisons with related species, we find that our reference genome is among the most complete marsupial assemblies currently available. Using this assembly, we infer the species demographic history and identify potential evidence of a long-term decline beginning in the late Pleistocene. Finally, we identify a deletion at the ASIP locus that likely drives differences in pelage color between the eastern quoll and the closely related Tasmanian devil (Sarcophilus harrisii). The genomic resources we present are valuable new tools for evolutionary and conservation genomic studies.

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↗

bioRxiv2022

The complete sequence of a human Y chromosome

The human Y chromosome has been notoriously difficult to sequence and assemble because of its complex repeat structure including long palindromes, tandem repeats, and segmental duplications1-3. As a result, more than half of the Y chromosome is missing from the GRCh38 reference sequence and it remains the last human chromosome to be finished4, 5. Here, the Telomere-to-Telomere (T2T) consortium presents the complete 62,460,029 base pair sequence of a human Y chromosome from the HG002 genome (T2T-Y) that corrects multiple errors in GRCh38-Y and adds over 30 million base pairs of sequence to the reference, revealing the complete ampliconic structures of TSPY, DAZ, and RBMY gene families; 41 additional protein-coding genes, mostly from the TSPY family; and an alternating pattern of human satellite 1 and 3 blocks in the heterochromatic Yq12 region. We have combined T2T-Y with a prior assembly of the CHM13 genome4 and mapped available population variation, clinical variants, and functional genomics data to produce a complete and comprehensive reference sequence for all 24 human chromosomes.

genomics↗

bioRxiv2021

Complete genomic and epigenetic maps of human centromeres

Existing human genome assemblies have almost entirely excluded highly repetitive sequences within and near centromeres, limiting our understanding of their sequence, evolution, and essential role in chromosome segregation. Here, we present an extensive study of newly assembled peri/centromeric sequences representing 6.2% (189.9 Mb) of the first complete, telomere-to-telomere human genome assembly (T2T-CHM13). We discovered novel patterns of peri/centromeric repeat organization, variation, and evolution at both large and small length scales. We also found that inner kinetochore proteins tend to overlap the most recently duplicated subregions within centromeres. Finally, we compared chromosome X centromeres across a diverse panel of individuals and uncovered structural, epigenetic, and sequence variation at single-base resolution across these regions. In total, this work provides an unprecedented atlas of human centromeres to guide future studies of their complex and critical functions as well as their unique evolutionary dynamics. One-sentence summaryDeep characterization of fully assembled human centromeres reveals their architecture and fine-scale organization, variation, and evolution.

genomics↗

bioRxiv2021

From telomere to telomere: the transcriptional and epigenetic state of human repeat elements

Mobile elements and highly repetitive genomic regions are potent sources of lineage-specific genomic innovation and fingerprint individual genomes. Comprehensive analyses of large, composite or arrayed repeat elements and those found in more complex regions of the genome require a complete, linear genome assembly. Here we present the first de novo repeat discovery and annotation of a complete human reference genome, T2T-CHM13v1.0. We identified novel satellite arrays, expanded the catalog of variants and families for known repeats and mobile elements, characterized new classes of complex, composite repeats, and provided comprehensive annotations of retroelement transduction events. Utilizing PRO-seq to detect nascent transcription and nanopore sequencing to delineate CpG methylation profiles, we defined the structure of transcriptionally active retroelements in humans, including for the first time those found in centromeres. Together, these data provide expanded insight into the diversity, distribution and evolution of repetitive regions that have shaped the human genome.

genomics↗

bioRxiv2021

The complete sequence of a human genome

In 2001, Celera Genomics and the International Human Genome Sequencing Consortium published their initial drafts of the human genome, which revolutionized the field of genomics. While these drafts and the updates that followed effectively covered the euchromatic fraction of the genome, the heterochromatin and many other complex regions were left unfinished or erroneous. Addressing this remaining 8% of the genome, the Telomere-to-Telomere (T2T) Consortium has finished the first truly complete 3.055 billion base pair (bp) sequence of a human genome, representing the largest improvement to the human reference genome since its initial release. The new T2T-CHM13 reference includes gapless assemblies for all 22 autosomes plus Chromosome X, corrects numerous errors, and introduces nearly 200 million bp of novel sequence containing 2,226 paralogous gene copies, 115 of which are predicted to be protein coding. The newly completed regions include all centromeric satellite arrays and the short arms of all five acrocentric chromosomes, unlocking these complex regions of the genome to variational and functional studies for the first time.

genomics↗