Machine and Deep Learning Methods for Predicting 3D Genome Organization.

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.

Acquisition and Analysis Methods for Hi-C Data from Medaka Early Embryos.

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.

Analysis and Visualization of Multiple Hi-C and Micro-C Data with CustardPy.

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.

Integrative Modeling of 3D Genome Organization by Bayesian Molecular Dynamics Simulations with Hi-C Metainference.

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.

4D Genome Analysis Using PHi-C2.

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.

Genome Annotation.

The hallmark of genome sequencing projects is to provide genetic information on a species with functional annotations of genes and proteins. This process heavily relies on genome annotation based on homology detections from previously known genomic data. The rapid advancement of genome sequencing technologies has made genome sequencing affordable and effective in terms of the time frame for the generation of genomic data. Hence, genome sequencing has become a common practice. The annotation and characterization of newly sequenced genomes are crucial factors for the success of any biological experiment based on genomic data. The proteogenomic sector requires annotated genome further characterization of proteomic-based studies, and these are coupled with genomic and RNA-seq data. This chapter describes the genome annotation process from scratch genome sequencing to general genome annotation and specialized genome annotation using BLAST, BLAT2GO (now OMICSBOX), PANNZER, gene ontology (GO), and KEGG. It also covers different processes like repeat identification and masking, gene prediction, genome-wide annotation process, and RNA-seq protocol. It also focuses on genes of interest such as genes associated with BGCs (biosynthetic gene clusters), carbohydrate-active enzymes (CAZymes), serpins (serine protease inhibitors), membrane transporters, and toxins. Manual annotation is also a critical step for at least some groups of genes, which are often critical for the species in consideration. This chapter also briefly describes the phylogenetic and phylogenomic processes required during genome annotation.

The origin and maintenance of supergenes contributing to ecological adaptation in Atlantic herring.

Chromosomal inversions are associated with local adaptation in many species. However, questions regarding how they are formed, maintained and impact various other evolutionary processes remain elusive. Here, using a large genomic dataset of long-read and short-read sequencing, we ask these questions in one of the most abundant vertebrates on Earth, the Atlantic herring. This species has four megabase-sized inversions associated with ecological adaptation that correlate with water temperature. The S and N inversion alleles at these four loci dominate in the southern and northern parts, respectively, of the species distribution in the North Atlantic Ocean. By determining breakpoint coordinates of the four inversions and the structural variations surrounding them, we hypothesize that these inversions are formed by ectopic recombination between duplicated sequences immediately outside of the inversions. We show that these are old inversions (>1 MY), albeit formed after the split between the Atlantic herring and its sister species, the Pacific herring. There is evidence for extensive gene flux between inversion alleles at all four loci. The large Ne of herring combined with the common occurrence of opposite homozygotes across the species distribution has allowed effective purifying selection to prevent the accumulation of genetic load and repeats within the inversions.

DNA methylation analysis to differentiate reference, breed, and parent-of-origin effects in the bovine pangenome era.

BACKGROUND: Most DNA methylation studies have used a single reference genome with little attention paid to the bias introduced due to the reference chosen. Reference genome artifacts and genetic variation, including single nucleotide polymorphisms (SNPs) and structural variants (SVs), can lead to differences in methylation sites (CpGs) between individuals of the same species. We analyzed whole-genome bisulfite sequencing data from the fetal liver of Angus (Bos taurus taurus), Brahman (Bos taurus indicus), and reciprocally crossed samples. Using reference genomes for each breed from the Bovine Pangenome Consortium, we investigated the influence of reference genome choice on the breed and parent-of-origin effects in methylome analyses. RESULTS: Our findings revealed that ∼75% of CpG sites were shared between Angus and Brahman, ∼5% were breed specific, and ∼20% were unresolved. We demonstrated up to ∼2% quantification bias in global methylation when an incorrect reference genome was used. Furthermore, we found that SNPs impacted CpGs 13 times more than other autosomal sites (P < $5 \times {10}^{ - 324}$) and SVs contained 1.18 times (P < $5 \times {10}^{ - 324}$) more CpGs than non-SVs. We found a poor overlap between differentially methylated regions (DMRs) and differentially expressed genes (DEGs) and suggest that DMRs may be impacting enhancers that target these DEGs. DMRs overlapped with imprinted genes, of which 1, DGAT1, which is important for fat metabolism and weight gain, was found in the breed-specific and sire-of-origin comparisons. CONCLUSIONS: This work demonstrates the need to consider reference genome effects to explore genetic and epigenetic differences accurately and identify DMRs involved in controlling certain genes.

Genomic exploration of the endangered oriental stork, Ciconia boyciana, sheds light on migration adaptation and future conservation.

BACKGROUND: The oriental stork, Ciconia boyciana, is an endangered migratory bird listed on the International Union for Conservation of Nature's Red List. The bird population has experienced a rapid decline in the past decades, with nest locations and stop-over sites largely degraded due to human-bird conflicts. Multipronged conservation efforts are required to secure the future of oriental storks. We propose that a thorough understanding of the genome-wide genetic background of this threatened bird species is critical to make future conservation strategies. FINDINGS: In this study, the first chromosome-scale reference genome was presented for the oriental stork with high quality, contiguity, and accuracy. The assembled genome size was 1.24 Gb with a scaffold N50 of 103 Mb, and 1.23 Gb contigs (99.32%) were anchored to 35 chromosomes. Population genomic analysis did not show a genetic structure in the wild population. Genome-wide genetic diversity (π = 0.0012) of the oriental stork was at a moderate to high level among threatened bird species, and the inbreeding risk was also not significant (FROH = 5.56% ± 5.30%). Reconstruction of demographic history indicated a rapid recent population decline likely driven by human activities. Genes that were under positive selection associated with the migratory trait were identified in relation to the long-term potentiation, photoreceptor cell organization, circadian rhythm, muscle development, and energy metabolism, indicating the essential interplay between genetic and ecological adaptation. CONCLUSIONS: Our study presents the first chromosome-scale genome assembly of the oriental stork and provides a genomic basis for understanding a genetic background of the oriental stork, the population's extinction risks, and the migratory characteristics, which will facilitate the decision of future conservation plans for this species.

Identification of sex-specific markers using genome re-sequencing in the blunt snout bream (Megalobrama amblycephala).

BACKGROUND: The blunt snout bream (Megalobrama amblycephala) is an important economic freshwater fish in China with tender flesh and high nutritional value. With the cultivation of superior new varieties and the expansion of breeding scale, it becomes imperative to employ sex-control technology to cultivate monosexual populations of M. amblycephala, thereby preventing the deterioration of desirable traits. The development of specific markers capable of accurately identifying the sex of M. amblycephala would facilitate the determination of the genetic sex of the breeding population before gonad maturation, thereby expediting the processes of sex-controlled breeding of M. amblycephala. RESULTS: A whole-genome re-sequencing was performed for 116 females and 141 males M. amblycephala collected from nine populations. Seven candidate male-specific sequences were identified through comparative analysis of male and female genomes, which were further compared with the sequencing data of 257 individuals, and finally three male-specific sequences were generated. These three sequences were further validated by PCR amplification in 32 males and 32 females to confirm their potential as male-specific molecular markers for M. amblycephala. One of these markers showed potential applicability in M. pellegrini as well, enabling males to be identified using this specific molecular marker. CONCLUSIONS: The study provides a high-efficiency and cost-effective approach for the genetic sex identification in two species of Megalobrama. The developed markers in this study have great potential in facilitating sex-controlled breeding of M. amblycephala and M. pellegrini, while also contributing valuable insights into the underlying mechanisms of fish sex determination.

Genomic Sequencing to Detect Cross-Breeding Quality in Dogs: An Example Studying Disorders in Sexual Development.

Disorders of sexual development (DSDs) in dogs, similar to humans, arise from genetic mutations, gonadal differentiation, or phenotypic sex development. The French Bulldog, a breed that has seen a surge in popularity and demand, has also shown a marked increase in DSD incidence. This study aims to characterize the genetic underpinnings of DSDs in a French Bulldog named Brutus, exhibiting ambiguous genitalia and internal sexual anatomy, and to explore the impact of breeding practices on genetic diversity within the breed. We utilized a comprehensive approach combining conventional cytogenetics, molecular techniques, and deep sequencing to investigate the genetic profile of Brutus. The sequence data were compared to three other male French Bulldogs' genome sequences with typical reproductive anatomy, including Brutus's father and the canine reference genome (CanFam6). We found a Robertsonian fusion involving chromosome 23 previously reported in dogs as a causative mutation responsible for sex reversal syndrome. Our findings revealed a 22% mosaicism (78,XX/77,XX), the absence of the sex-determining region (SRY) gene, and the presence of 43 unique Single Nucleotide Variants (SNVs) not inherited from the father. Notably, the run of homozygosity (ROH) analysis showed Brutus has a higher number of homozygous segments compared to other Bulldogs, with a total length of these fragments 50% greater than the average, strongly suggesting this dog is the product of the mating between siblings. Although no direct causative genes for the DSD phenotype were identified, four candidate loci warrant further investigation. Our study highlighted the need for a better annotated and curated reference dog genome to define genes causative of any specific phenotype, suggests a potential genetic basis for the DSD phenotype in dogs, and underscores the consequences of uncontrolled breeding practices in French Bulldogs. These findings highlight the importance of implementing strategic genetic management to preserve genetic health and diversity in canine populations.

Genome-Wide Identification and Expression Analysis of the BTB Gene Superfamily Provides Insight into Sex Determination and Early Gonadal Development of Alligator sinensis.

The BTB gene superfamily is widely distributed among higher eukaryotes and plays a significant role in numerous biological processes. However, there is limited knowledge about the structure and function of BTB genes in the critically endangered species Alligator sinensis, which is endemic to China. A total of 170 BTB genes were identified from the A. sinensis genome, classified into 13 families, and unevenly distributed across 16 chromosomes. Analysis of gene duplication events yielded eight pairs of tandem duplication genes and six pairs of segmental duplication genes. Phylogenetics shows that the AsBTB genes are evolutionarily conserved. The cis-regulatory elements in the AsBTB family promoter region reveal their involvement in multiple biological processes. Protein interaction network analysis indicates that the protein interactions of the AsBTB genes are centered around CLU-3, mainly participating in the regulation of biological processes through the ubiquitination pathway. The expression profile and protein interaction network analysis of AsBTB genes during sex differentiation and early gonadal development indicate that AsBTB genes are widely expressed in this process and involves numerous genes and pathways for regulation. This study provides a basis for further investigation of the role of the BTB gene in sex differentiation and gonadal development in A. sinensis.

Comprehensive evaluation and guidance of structural variation detection tools in chicken whole genome sequence data.

BACKGROUND: Structural variations (SVs) are widespread across genome and have a great impact on evolution, disease, and phenotypic diversity. Despite the development of numerous bioinformatic tools, commonly referred to as SV callers, tailored for detecting SVs using whole genome sequence (WGS) data and employing diverse algorithms, their performance necessitates rigorous evaluation with real data and validated SVs. Moreover, a considerable proportion of these tools have been primarily designed and optimized using human genome data. Consequently, their applicability and performance in Avian species, characterized by smaller genomes and distinct genomic architectures, remain inadequately assessed. RESULTS: We performed a comprehensive assessment of the performance of ten widely used SV callers using population-level real genomic data with the validated five common types of SVs. The performance of SV callers varies with the types and sizes of SVs. As compared with other tools, GRIDSS, Lumpy, Wham, and Manta present better detection accuracy. Pindel can detect more small SVs than others. CNVnator and CNVkit can detect more medium and large copy number variations. Given the poor consistency among different SV callers, the combination calling strategy is not recommended. All tools show poor ability in the detection of insertions (especially with size > 150 bp). At least 50× read depth is required to detect more than 80% of the SVs for most tools. CONCLUSIONS: This study highlights the importance and necessity of using real sequencing data, rather than simulated data only, with validated SVs for SV caller evaluation. Some practical guidance and suggestions are provided for SV detection in future researches.

The loach haplotype-resolved genome and the identification of Mex3a involved in fish air breathing.

Fish air breathing is crucial for the transition of vertebrates from water to land. So far, the genes involved in fish air breathing have not been well identified. Here, we performed gene enrichment analysis of positively selected genes (PSGs) in loach (Misgurnus anguillicaudatus, an air-breathing fish) in comparison to Triplophysa tibetana (a non-air-breathing fish), haplotype-resolved genome assembly of the loach, and gene evolutionary analysis of air-breathing and non-air-breathing fishes and found that the PSG mex3a originated from ancient air-breathing fish species. Deletion of Mex3a impaired loach air-breathing capacity by inhibiting angiogenesis through its interaction with T-box transcription factor 20. Mex3a overexpression significantly promoted angiogenesis. Structural analysis and point mutation revealed the critical role of the 201st amino acid in loach Mex3a for angiogenesis. Our findings innovatively indicate that the ancient mex3a is a fish air-breathing gene, which holds significance for understanding fish air breathing and provides a valuable resource for cultivating hypoxia-tolerant fish varieties.

Genome-wide comparative analyses for selection signatures indicate candidate genes for between-breed variability in copper accretion in sheep.

The problem of copper (Cu) intoxication and deficiency continues to impact economic gains and animal welfare in sheep husbandry. This study investigated the ovine genome for regions and potential genes under selection for Cu accretion between sheep breeds. For this, we compared ovine single nucleotide polymorphism (SNP) data of three Cu-susceptible breeds with three Cu-tolerant breeds. After merging SNP data of breeds and removal of related individuals, a total of 229 sheep and 45 640 autosomal SNPs were left. Then, we selected 14 individuals per breed into two datasets (datasets 1 and 2) for analysis of selection signatures using the Fixation index, cross-population extended haplotype homozygosity and haplotype-based FLK methods. Selection regions shared by both datasets detected by at least two methods revealed regions on OAR 4, 8 and 11 containing 54 candidate genes under selection for Cu accretion. Enrichment analysis revealed that 19 gene ontologies and 1 enriched Kyoto encyclopaedia of genes and genomes pathway terms were associated with the candidate genes under selection. Genes such as TP53, TNFSF13, TNFSF12, ALOX15, ALOX12, EIF5A and PREP are associated with the regulation of Cu homeostasis, programmed cell death or inflammatory response. We also found an enrichment of arachidonate 15-lipoxygenase activity, arachidonate 12-lipoxygenase activity and ferroptosis that influence cellular inflammation and cell death. These results shed light on ovine genomic regions under selection for Cu accretion and provide information on candidate genes for further studies on breed differences in ovine Cu accretion.

Chromosome-level genome assembly of the cashmere goat.

The goat, an early domesticated ruminant, is a reliable source of cashmere, meat and milk in global agricultural production. Despite this, the genome of cashmere-rich goats has yet to be characterized. Here, we assembled the nearly complete genome of a cashmere goat from a highly economically valuable Inner Mongolian Cashmere buck, utilizing a combination of PacBio HiFi, ONT ultra-long reads, and Hi-C technologies. The size of this genome is 2.76 Gb, with a contig N50 of 95.22 Mb. All assembled sequences were anchored onto 29 autosomes and both sex chromosomes, with only two gaps present on the X chromosome. We identified 1,333.29 Mb (48.26%) of repetitive sequences and predicted 22,480 protein-coding genes. Assembly quality assessment of the genome demonstrated that our assembled cashmere goat genome surpasses the continuity, completeness, and accuracy of other published goat genomes. Taken together, we provided the first cashmere goat assembly, bridging the gap in the genome of important economic breeds of domestic goats, and providing a valuable reference resource for goat genetics and genome research.

Chromosome-level genome provides insights into evolution and diving adaptability in the vulnerable common pochard (Aythya ferina).

The common pochard (Aythya ferina) is a freshwater diving duck found in the Palearctic region that has been classified as vulnerable by the IUCN due to continuous and rapid population declines across their distribution. To gain a better understanding of its genetic mechanism of adaptive evolution, we successfully sequenced and assembled the first high-quality chromosome-level genome of A. ferina using Illumina, Nanopore and Hi-C sequencing technologies. A total assembly length of 1,130.78 Mbp was obtained, with over 98.81% (1,117.37Mbp) of sequence anchored to 35 pseudo-chromosomes. We predicted 17,232 protein-coding genes, 95.9% of which were functionally annotated. We identified 339 expanded and 937 contracted gene families in the genome of A. ferina, and detected 95 genes that have been positively selected. The significantly enriched Gene Ontology and enriched pathways were related to energy metabolism, immune, nervous, and sensory systems, suggests that these factors likely played an important role in its evolution. Importantly, we recovered signatures of positive selection on genes related to vasoconstriction that may be associated with thermoregulatory adaptations of A. ferina for underwater diving. Overall, the high-quality genome assembly and annotation in this study provides valuable genomic resources for ecological and evolutionary studies, as well as toward the conservation of A. ferina.

A chromosome-level genome assembly of Cape hare (Lepus capensis).

The Cape hare (Lepus capensis) is among the most widely distributed hare species globally, inhabiting extensive regions across Africa, the Middle East, and Central Asia. However, evolutionary and genetic research on L. capensis was seriously impeded by the absence of a reference genome. Here, we assembled and constructed a chromosome-level genome of L. capensis (with scaffolds anchored to 25 chromosomes and a total assembled length of 2.9 Gb, achieving a contig N50 length of 124.44 Mb) using PacBio HiFi sequencing and Hi-C assembly technology. Evaluation using BUSCO indicated the genome assembly to be 98.2% complete. The de novo prediction revealed that repetitive sequences constitute 46.13% of the entire genome, and long interspersed nuclear elements (LINEs) constituted the largest portion. We annotated a total of 13, 868 protein-coding genes using transcriptomes from two tissues (muscle and skin). This high-quality reference genome serves as a valuable genomic resource for advancing genetic studies in this species.

Chromosome-level genome assembly of the sacoglossan sea slug Elysia timida (Risso, 1818).

BACKGROUND: Sequencing and annotating genomes of non-model organisms helps to understand genome architecture, the genetic processes underlying species traits, and how these genes have evolved in closely-related taxa, among many other biological processes. However, many metazoan groups, such as the extremely diverse molluscs, are still underrepresented in the number of sequenced and annotated genomes. Although sequencing techniques have recently improved in quality and quantity, molluscs are still neglected due to difficulties in applying standardized protocols for obtaining genomic data. RESULTS: In this study, we present the chromosome-level genome assembly and annotation of the sacoglossan sea slug species Elysia timida, known for its ability to store the chloroplasts of its food algae. In particular, by optimizing the long-read and chromosome conformation capture library preparations, the genome assembly was performed using PacBio HiFi and Arima HiC data. The scaffold and contig N50s, at 41.8 Mb and 1.92 Mb, respectively, are approximately 30-fold and fourfold higher compared to other published sacoglossan genome assemblies. Structural annotation resulted in 19,904 protein-coding genes, which are more contiguous and complete compared to publicly available annotations of Sacoglossa with respect to metazoan BUSCOs. We found no evidence for horizontal gene transfer (HGT), i.e. no photosynthetic genes encoded in the sacoglossan nucleus genome. However, we detected genes encoding polyketide synthases in E. timida, indicating that polypropionates are produced. HPLC-MS/MS analysis confirmed the presence of a large number of polypropionates, including known and yet uncharacterised compounds. CONCLUSIONS: We can show that our methodological approach helps to obtain a high-quality genome assembly even for a "difficult-to-sequence" organism, which may facilitate genome sequencing in molluscs. This will enable a better understanding of complex biological processes in molluscs, such as functional kleptoplasty in Sacoglossa, by significantly improving the quality of genome assemblies and annotations.

Genomic adaptation to small population size and saltwater consumption in the critically endangered Cat Ba langur.

Many mammal species have declining populations, but the consequences of small population size on the genomic makeup of species remain largely unknown. We investigated the evolutionary history, genetic load and adaptive potential of the Cat Ba langur (Trachypithecus poliocephalus), a primate species endemic to Vietnam's famous Ha Long Bay and with less than 100 living individuals one of the most threatened primates in the world. Using high-coverage whole genome data of four wild individuals, we revealed the Cat Ba langur as sister species to its conspecifics of the northern limestone langur clade and found no evidence for extensive secondary gene flow after their initial separation. Compared to other primates and mammals, the Cat Ba langur showed low levels of genetic diversity, long runs of homozygosity, high levels of inbreeding and an excess of deleterious mutations in homozygous state. On the other hand, genetic diversity has been maintained in protein-coding genes and on the gene-rich human chromosome 19 ortholog, suggesting that the Cat Ba langur retained most of its adaptive potential. The Cat Ba langur also exhibits several unique non-synonymous variants that are related to calcium and sodium metabolism, which may have improved adaptation to high calcium intake and saltwater consumption.