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The three-dimensional (3D) structure of the genome undergoes dynamic changes during the developmental process. While Hi-C is a technique that enables the acquisition of genome 3D structure data across various species and cell types, existing Hi-C analysis programs may face challenges in detecting and comparing structures effectively depending on the characteristics of the genome or cell type. Here, we describe a method for acquiring Hi-C data from medaka early embryos and quantifying the structural changes during the developmental process.

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Three-dimensional (3D) genome structure plays crucial roles in biological processes and disease pathogenesis. Hi-C and Micro-C, well-established methods for 3D genome analysis, can identify a variety of 3D genome structures. However, selecting appropriate pipelines and tools for the analysis and setting up the required computing environment can sometimes pose challenges. To address this, we have introduced CustardPy, a Docker-based pipeline specifically designed for 3D genome analysis. CustardPy is designed to compare and evaluate multiple samples and wraps several existing tools to cover the entire workflow from FASTQ mapping to visualization. In this chapter, we demonstrate how to analyze and visualize Hi-C data using CustardPy and introduce several 3D genome features observed in Hi-C data.

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Polymer modeling has been playing an increasingly important role in complementing 3D genome experiments, both to aid their interpretation and to reveal the underlying molecular mechanisms. This chapter illustrates an application of Hi-C metainference, a Bayesian approach to explore the 3D organization of a target genomic region by integrating experimental contact frequencies into a prior model of chromatin. The method reconstructs the conformational ensemble of the target locus by combining molecular dynamics simulation and Monte Carlo sampling from the posterior probability distribution given the data. Using prior chromatin models at both 1 kb and nucleosome resolution, we apply this approach to a 30 kb locus of mouse embryonic stem cells consisting of two well-defined domains linking several gene promoters together. Retaining the advantages of both physics-based and data-driven strategies, Hi-C metainference can provide an experimentally consistent representation of the system while at the same time retaining molecular details necessary to derive physical insights.

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Hi-C methods reveal 3D genome features but lack correspondence to dynamic chromatin behavior. PHi-C2, Python software, addresses this gap by transforming Hi-C data into polymer models. After the optimization algorithm, it enables us to calculate 3D conformations and conduct dynamic simulations, providing insights into chromatin dynamics, including the mean-squared displacement and rheological properties. This chapter introduces PHi-C2 usage, offering a tutorial for comprehensive 4D genome analysis.

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Three-dimensional (3D) chromatin interactions, such as enhancer-promoter interactions (EPIs), loops, topologically associating domains (TADs), and A/B compartments, play critical roles in a wide range of cellular processes by regulating gene expression. Recent development of chromatin conformation capture technologies has enabled genome-wide profiling of various 3D structures, even with single cells. However, current catalogs of 3D structures remain incomplete and unreliable due to differences in technology, tools, and low data resolution. Machine learning methods have emerged as an alternative to obtain missing 3D interactions and/or improve resolution. Such methods frequently use genome annotation data (ChIP-seq, DNAse-seq, etc.), DNA sequencing information (k-mers and transcription factor binding site (TFBS) motifs), and other genomic properties to learn the associations between genomic features and chromatin interactions. In this review, we discuss computational tools for predicting three types of 3D interactions (EPIs, chromatin interactions, and TAD boundaries) and analyze their pros and cons. We also point out obstacles to the computational prediction of 3D interactions and suggest future research directions.

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We introduce XGR-model (or XGRm), a web server made accessible at http://www.xgrm.pro, with the aim of meeting the increasing demand for effectively interpreting summary-level genomic data in model organisms. Currently, it hosts two enrichment analysers and two subnetwork analysers to support enrichment and subnetwork analyses for user-input mouse genomic data, whether gene-centric or genomic region-centric. The enrichment analysers identify ontology term enrichments for input genes (GElyser) or for genes linked from input genomic regions (RElyser). The subnetwork analysers rely on our previously established network algorithm to identify gene subnetworks from input gene-centric summary data (GSlyser) or from input region-centric summary data (RSlyser), leveraging network information about either functional interactions or pathway-derived interactions. Collectively, XGRm offers an all-in-one solution for gaining systems biology insights into summary-level genomic data in mice, underpinned by our commitment to regular updates as well as natural extensions to other model organisms.

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BACKGROUND: The Zic family of transcription factors (TFs) promote both proliferation and maturation of cerebellar granule neurons (CGNs), raising the question of how a single, constitutively expressed TF family can support distinct developmental processes. Here we use an integrative experimental and bioinformatic approach to discover the regulatory relationship between Zic TF binding and changing programs of gene transcription during postnatal CGN differentiation. RESULTS: We first established a bioinformatic pipeline to integrate Zic ChIP-seq data from the developing mouse cerebellum with other genomic datasets from the same tissue. In newborn CGNs, Zic TF binding predominates at active enhancers that are co-bound by developmentally regulated TFs including Atoh1, whereas in mature CGNs, Zic TF binding consolidates toward promoters where it co-localizes with activity-regulated TFs. We then performed CUT&RUN-seq in differentiating CGNs to define both the time course of developmental shifts in Zic TF binding and their relationship to gene expression. Mapping Zic TF binding sites to genes using chromatin looping, we identified the set of Zic target genes that have altered expression in RNA-seq from Zic1 or Zic2 knockdown CGNs. CONCLUSIONS: Our data show that Zic TFs are required for both induction and repression of distinct, developmentally regulated target genes through a mechanism that is largely independent of changes in Zic TF binding. We suggest that the differential collaboration of Zic TFs with other TF families underlies the shift in their biological functions across CGN development.

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BACKGROUND: The Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis, YFP) and the East Asian finless porpoise (Neophocaena asiaeorientalis sunameri, EFP) are 2 subspecies of the narrow-ridged finless porpoise that live in freshwater and saltwater, respectively. The main objective of this study was to provide contiguous chromosome-level genome assemblies for YFP and EFP. RESULTS: Here, we generated and upgraded the genomes of YFP and EFP at the telomere-to-telomere level through the integration of PacBio HiFi long reads, ultra-long ONT reads, and Hi-C sequencing data with a total size of 2.48 Gb and 2.50 Gb, respectively. The scaffold N50 of 2 genomes was 125.12 Mb (YFP) and 128 Mb (EFP) with 1 contig for 1 chromosome. The telomere repeat and centromere position were clearly identified in both YFP and EFP genomes. In total, 5,480 newfound genes were detected in the YFP genome, including 56 genes located in the newly identified centromere regions. Additionally, synteny blocks, structural similarities, phylogenetic relationships, gene family expansion, and inference of selection were studied in connection with the genomes of other related mammals. CONCLUSIONS: Our research findings provide evidence for the gradual adaptation of EFP in a marine environment and the potential sensitivity of YFP to genetic damage. Compared to the 34 cetacean genomes sourced from public databases, the 2 new assemblies demonstrate superior continuity with the longest contig N50 and scaffold N50 values, as well as the lowest number of contigs. The improvement of telomere-to-telomere gap-free reference genome resources supports conservation genetics and population management for finless porpoises.

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The muskox (Ovibos moschatus), an integral component and iconic symbol of arctic biocultural diversity, is under threat by rapid environmental disruptions from climate change. We report a chromosomal-level haploid genome assembly of a muskox from Banks Island in the Canadian Arctic Archipelago. The assembly has a contig N50 of 44.7 Mbp, a scaffold N50 of 112.3 Mbp, a complete representation (100%) of the BUSCO v5.2.2 set of 9225 mammalian marker genes and is anchored to the 24 chromosomes of the muskox. Tabulation of heterozygous single nucleotide variants in our specimen revealed a very low level of genetic diversity, which is consistent with recent reports of the muskox having the lowest genome-wide heterozygosity among the ungulates. While muskox populations are currently showing no overt signs of inbreeding depression, environmental disruptions are expected to strain the genomic resilience of the species. One notable impact of rapid climate change in the Arctic is the spread of emerging infectious and parasitic diseases in the muskox, as exemplified by the range expansion of muskox lungworms, and the recent fatal outbreaks of Erysipelothrix rhusiopathiae, a pathogen normally associated with domestic swine and poultry. As a genomics resource for conservation management of the muskox against existing and emerging disease modalities, we annotated the genes of the major histocompatibility complex on chromosome 2 and performed an initial assessment of the genetic diversity of this complex. This resource is further supported by the annotation of the principal genes of the innate immunity system, genes that are rapidly evolving and under positive selection in the muskox, genes associated with environmental adaptations, and the genes associated with socioeconomic benefits for Arctic communities such as wool (qiviut) attributes. These annotations will benefit muskox management and conservation.

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The Harpy Eagle (Harpia harpyja) is an iconic species that inhabits forested landscapes in Neotropical regions, with decreasing population trends mainly due to habitat loss, and currently classified as vulnerable. Here, we report on a chromosome-scale genome assembly for a female individual combining long reads, optical mapping, and chromatin conformation capture reads. The final assembly spans 1.35 Gb, with N50scaffold equal to 58.1 Mb and BUSCO completeness of 99.7%. We built the first extensive transposable element (TE) library for the Accipitridae to date and identified 7,228 intact TEs. We found a burst of an unknown TE ~ 13-22 million years ago (MYA), coincident with the split of the Harpy Eagle from other Harpiinae eagles. We also report a burst of solo-LTRs and CR1 retrotransposons ~ 31-33 MYA, overlapping with the split of the ancestor to all Harpiinae from other Accipitridae subfamilies. Comparative genomics with other Accipitridae, the closely related Cathartidae and Galloanserae revealed major chromosome-level rearrangements at the basal Accipitriformes genome, in contrast to a conserved ancient genome architecture for the latter two groups. A historical demography reconstruction showed a rapid decline in effective population size over the last 20,000 years. This reference genome serves as a crucial resource for future conservation efforts towards the Harpy Eagle.

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The polar bear (Ursus maritimus) occupies a relatively narrow ecological niche, with many traits adapted for cold temperatures, movement across snow, ice and open water, and for consuming highly lipid-dense prey species. The divergence of polar bears from brown bears (Ursus arctos) and their adaptation to their Arctic lifestyle is a well-known example of rapid evolution. Previous research investigating whole genomes uncovered twelve key genes that are highly differentiated between polar and brown bears, show signatures of selection in the polar bear lineage, and are associated with polar bear adaptation to the Arctic environment. Further research suggested fixed derived alleles in these genes arose from selection on both standing variation and de novo mutations in the evolution of polar bears. Here, we reevaluate these findings based on a larger and geographically more representative dataset of 119 polar bears and 135 brown bears, and assess the timing of derived allele fixation in polar bears by incorporating the genomes of two Late Pleistocene individuals (aged 130-100,000 years old and 100-70,000 years old). In contrast with previous results, we found no evidence of derived alleles fixed in present-day polar bears within the key genes arising from de novo mutation. Most derived alleles fixed in present-day polar bears were also fixed in the Late Pleistocene polar bears, suggesting selection occurred prior to 70,000 years ago. However, some derived alleles fixed in present-day polar bears were not fixed in the two Late Pleistocene polar bears, including at sites within APOB, LYST, and TTN. These three genes are associated with cardiovascular function, metabolism, and pigmentation, suggesting selection may have acted on different loci at different times.

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Disease can act as a driving force in shaping genetic makeup across populations, even species, if the impacts influence a particularly sensitive part of their life cycles. White-nose disease is caused by a fungal pathogen infecting bats during hibernation. The mycosis has caused massive population declines of susceptible species in North America, particularly in the genus Myotis. However, Myotis bats appear to tolerate infection in Eurasia, where the fungal pathogen has co-evolved with its bat hosts for an extended period of time. Therefore, with susceptible and tolerant populations, the fungal disease provides a unique opportunity to tease apart factors contributing to tolerance at a genomic level to and gain an understanding of the evolution of non-harmful in host-parasite interactions. To investigate if the fungal disease has caused adaptation on a genomic level in Eurasian bat species, we adopted both whole-genome sequencing approaches and a literature search to compile a set of 300 genes from which to investigate signals of positive selection in genomes of 11 Eurasian bats at the codon-level. Our results indicate significant positive selection in 38 genes, many of which have a marked role in responses to infection. Our findings suggest that white-nose syndrome may have applied a significant selective pressure on Eurasian Myotis-bats in the past, which can contribute their survival in co-existence with the pathogen. Our findings provide an insight on the selective pressure pathogens afflict on their hosts using methodology that can be adapted to other host-pathogen study systems.

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OBJECTIVES: The two oyster species studied hold considerable economic importance for artisanal harvest (Crassostrea rhizophorae) and aquaculture (Crassostrea gasar). Their draft genomes will play an important role in the application of genomic methods such as RNAseq, population-based genomic scans aiming at addressing expression responses to pollution stress, adaptation to salinity and temperature variation, and will also permit investigating the genetic bases and enable marker-assisted selection of economically important traits like shell and mantle coloration and resistance to temperature and disease. DATA DESCRIPTION: The draft assembly size of Crassostrea gasar is 506 Mbp, and of Crassostrea rhizophorae is 584 Mbp with scaffolds N50 of 11,3 Mbp and 4,9 Mbp, respectively. The general masked bases by RepeatMasker in both genomes were highly similar using different datasets. The masked bases varied from 9.41% in C. gasar to 10.05% in C. rhizophorae and 42.85% in C. gasar to 44.44% in C. rhizophorae using Dfam and RepeatModeler datasets, respectively. Functional annotation with eggNog resulted in 34,693 annotated proteins in C. rhizophorae and 26,328 in C. gasar. BUSCO analysis shows that almost 99% of genes (5,295) are complete in relation to the mollusk orthologous genes dataset (mollusca_odb10).

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The integration of proteomics data with constraint-based reconstruction and analysis (COBRA) models plays a pivotal role in understanding the relationship between genotype and phenotype and bridges the gap between genome-level phenomena and functional adaptations. Integrating a generic genome-scale model with information on proteins enables generation of a context-specific metabolic model which improves the accuracy of model prediction. This review explores methodologies for incorporating proteomics data into genome-scale models. Available methods are grouped into four distinct categories based on their approach to integrate proteomics data and their depth of modeling. Within each category section various methods are introduced in chronological order of publication demonstrating the progress of this field. Furthermore, challenges and potential solutions to further progress are outlined, including the limited availability of appropriate in vitro data, experimental enzyme turnover rates, and the trade-off between model accuracy, computational tractability, and data scarcity. In conclusion, methods employing simpler approaches demand fewer kinetic and omics data, consequently leading to a less complex mathematical problem and reduced computational expenses. On the other hand, approaches that delve deeper into cellular mechanisms and aim to create detailed mathematical models necessitate more extensive kinetic and omics data, resulting in a more complex and computationally demanding problem. However, in some cases, this increased cost can be justified by the potential for more precise predictions.

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BACKGROUND: The evolution of extracellular matrix is tightly linked to the evolution of organogenesis in metazoans. Tenascins are extracellular matrix glycoproteins of chordates that participate in integrin-signaling and morphogenetic events. Single tenascins are encoded by invertebrate chordates, and multiple tenascin paralogs are found in vertebrates (designated tenascin-C, tenascin-R, tenascin-W and tenascin-X) yet, overall, the evolution of this family has remained unclear. RESULTS: This study examines the genomes of hemichordates, cephalochordates, tunicates, agnathans, cartilaginous fishes, lobe-finned fishes, ray-finned fishes and representative tetrapods to identify predicted tenascin proteins. We comprehensively assess their evolutionary relationships by sequence conservation, molecular phylogeny and examination of conservation of synteny of the encoding genes. The resulting new evolutionary model posits the origin of tenascin in an ancestral chordate, with tenascin-C-like and tenascin-R-like paralogs emerging after a whole genome duplication event in an ancestral vertebrate. Tenascin-X appeared following a second round of whole genome duplication in an ancestral gnathostome, most likely from duplication of the gene encoding the tenascin-R homolog. The fourth gene, encoding tenascin-W (also known as tenascin-N), apparently arose from a local duplication of tenascin-R. CONCLUSIONS: The diversity of tenascin paralogs observed in agnathans and gnathostomes has evolved through selective retention of novel genes that arose from a combination of whole genome and local duplication events. The evolutionary appearance of specific tenascin paralogs coincides with the appearance of vertebrate-specific cell and tissue types where the paralogs are abundantly expressed, such as the endocranium and facial skeleton (tenascin-C), an expanded central nervous system (tenascin-R), and bone (tenascin-W).

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Prime editor, an editing tool based on the CRISPR/Cas9 system, allows for all 12 types of nucleotide exchanges and arbitrary indels in genomic sequences without the need for inducing DNA double-strand breaks. Despite its flexibility and precision, prime editing efficiency is still low and hindered by various factors such as target sites, editing types, and the length of the primer binding site. In this study, we developed a prime editing system by incorporating an RNA motif at the 3' terminal of the pegRNA and integrating all twin prime editor factors into a single plasmid. These two strategies enhanced prime editing efficiency at target sites by up to 3.58-fold and 2.19-fold, respectively. Subsequently, enhanced prime editor was employed in goat cells and embryos to efficiently insert a 38 bp attB sequence into the Gt(ROSA)26Sor (Rosa26) and C-C motif chemokine receptor 5 (CCR5) loci. The enhanced prime editor can mediate 11.9% and 6.8% editing efficiency in parthenogenetic activation of embryos through embryo microinjection. In summary, our study introduces a modified prime editing system with improved editing and transfection efficiency, making it more suitable for inserting foreign sequences into primary cells and embryos. These results broaden the potential applications of prime editing technologies in the production of transgenic animals.

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Abalone (family Haliotidae) are an ecologically and economically significant group of marine gastropods that can be found in tropical and temperate waters. To date, only a few Haliotis genomes are available, all belonging to temperate species. Here, we provide the first chromosome-scale abalone genome assembly and the first reference genome of the tropical abalone Haliotis asinina. The combination of PacBio long-read HiFi sequencing and Dovetail's Omni-C sequencing allowed the chromosome-level assembly of this genome, while PacBio Isoform sequencing across five tissue types enabled the construction of high-quality gene models. This assembly resulted in 16 pseudo-chromosomes spanning over 1.12 Gb (98.1% of total scaffolds length), N50 of 67.09 Mb, the longest scaffold length of 105.96 Mb, and a BUSCO completeness score of 97.6%. This study identified 25,422 protein-coding genes and 61,149 transcripts. In an era of climate change and ocean warming, this genome of a heat-tolerant species can be used for comparative genomics with a focus on thermal resistance. This high-quality reference genome of H. asinina is a valuable resource for aquaculture, fisheries, and ecological studies.

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BACKGROUND: Studying the composition rules and evolution mechanisms of genome sequences are core issues in the post-genomic era, and k-mer spectrum analysis of genome sequences is an effective means to solve this problem. RESULT: We divided total 8-mers of genome sequences into 16 kinds of XY-type due to XY dinucleotides number in 8-mers. Previous works explored that the independent unimodal distributions observed only in three CG-type 8-mer spectra, while non-CG type 8-mer spectra have not the universal phenomenon from prokaryotes to eukaryotes. On this basis, we analyzed the distribution variation of non-CG type 8-mer spectra across 889 animal genome sequences. Following the evolutionary order of animals from primitive to more complex, we found that the spectrum distributions gradually transition from unimodal to tri-modal. The relative distance from the average frequency of each non-CG type 8-mers to the center frequency is different within a species and among different species. For the 8-mers contain CG dinucleotides, we further divided these into 16 subsets, where each 8-mer contains both CG and XY dinucleotides, called XY1_CG1 subsets. We found that the separability values of XY1_CG1 spectra are closely related to the evolution and specificity of animals. Considering the constraint of Chargaff's second parity rule, we finally obtained 10 separability values as the feature set to characterize the evolution state of genome sequences. In order to verify the rationality of the feature set, we used 14 common classification algorithms to perform binary classification tests. The results showed that the accuracy (Acc) ranged between 98.70% and 83.88% among birds, other vertebrates and mammals. CONCLUSION: We proposed a credible feature set to characterizes the evolution state of genomes and obtained satisfied results by the feature set on large scale classification of animals.

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Horseshoe bats (genus Rhinolophus, family Rhinolophidae) represent an important group within chiropteran phylogeny due to their distinctive traits, including constant high-frequency echolocation, rapid karyotype evolution, and unique immune system. Advances in evolutionary biology, supported by high-quality reference genomes and comprehensive whole-genome data, have significantly enhanced our understanding of species origins, speciation mechanisms, adaptive evolutionary processes, and phenotypic diversity. However, genomic research and understanding of the evolutionary patterns of Rhinolophus are severely constrained by limited data, with only a single published genome of R. ferrumequinum currently available. In this study, we constructed a high-quality chromosome-level reference genome for the intermediate horseshoe bat ( R. affinis). Comparative genomic analyses revealed potential genetic characteristics associated with virus tolerance in Rhinolophidae. Notably, we observed expansions in several immune-related gene families and identified various genes functionally associated with the SARS-CoV-2 signaling pathway, DNA repair, and apoptosis, which displayed signs of rapid evolution. In addition, we observed an expansion of the major histocompatibility complex class II (MHC-II) region and a higher copy number of the HLA- DQB2 gene in horseshoe bats compared to other chiropteran species. Based on whole-genome resequencing and population genomic analyses, we identified multiple candidate loci (e.g., GLI3) associated with variations in echolocation call frequency across R. affinis subspecies. This research not only expands our understanding of the genetic characteristics of the Rhinolophus genus but also establishes a valuable foundation for future research.

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