Micro-C Analysis Workflow Using Pairtools and Juicer.

Three-dimensional (3D) chromosome structures are closely related to various chromosomal functions, and deep analysis of the structures is crucial for the elucidation of the functions. In recent years, chromosome conformation capture (3C) techniques combined with next-generation sequencing analysis have been developed to comprehensively reveal 3D chromosome structures. Micro-C is one such method that can detect the structures at nucleosome resolution. In this chapter, I provide a basic method for Micro-C analysis. I present and discuss a series of data analyses ranging from mapping to basic downstream analyses, including loop detection.

Step-by-Step Protocol to Generate Hi-C Contact Maps Using the rfy_hic2 Pipeline.

The Hi-C method has emerged as an indispensable tool for analyzing the 3D organization of the genome, becoming increasingly accessible and frequently utilized in chromatin research. To effectively leverage 3D genomics data obtained through advanced technologies, it is crucial to understand what processes are undertaken and what aspects require special attention within the bioinformatics pipeline. This protocol aims to demystify the Hi-C data analysis process for field newcomers. In a step-by-step manner, we describe how to process Hi-C data, from the initial sequencing of the Hi-C library to the final visualization of Hi-C contact data as heatmaps. Each step of the analysis is clearly explained to ensure an understanding of the procedures and their objectives. By the end of this chapter, readers will be equipped with the knowledge to transform raw Hi-C reads into informative visual representations, facilitating a deeper comprehension of the spatial genomic structures critical to cellular functions.

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.

Improved simultaneous mapping of epigenetic features and 3D chromatin structure via ViCAR.

Methods to measure chromatin contacts at genomic regions bound by histone modifications or proteins are important tools to investigate chromatin organization. However, such methods do not capture the possible involvement of other epigenomic features such as G-quadruplex DNA secondary structures (G4s). To bridge this gap, we introduce ViCAR (viewpoint HiCAR), for the direct antibody-based capture of chromatin interactions at folded G4s. Through ViCAR, we showcase the first G4-3D interaction landscape. Using histone marks, we also demonstrate how ViCAR improves on earlier approaches yielding increased signal-to-noise. ViCAR is a practical and powerful tool to explore epigenetic marks and 3D genome interactomes.

Assembly, comparative analysis, and utilization of a single haplotype reference genome for soybean.

Cultivar Williams 82 has served as the reference genome for the soybean research community since 2008, but is known to have areas of genomic heterogeneity among different sub-lines. This work provides an updated assembly (version Wm82.a6) derived from a specific sub-line known as Wm82-ISU-01 (seeds available under USDA accession PI 704477). The genome was assembled using Pacific BioSciences HiFi reads and integrated into chromosomes using HiC. The 20 soybean chromosomes assembled into a genome of 1.01Gb, consisting of 36 contigs. The genome annotation identified 48 387 gene models, named in accordance with previous assembly versions Wm82.a2 and Wm82.a4. Comparisons of Wm82.a6 with other near-gapless assemblies of Williams 82 reveal large regions of genomic heterogeneity, including regions of differential introgression from the cultivar Kingwa within approximately 30 Mb and 25 Mb segments on chromosomes 03 and 07, respectively. Additionally, our analysis revealed a previously unknown large (>20 Mb) heterogeneous region in the pericentromeric region of chromosome 12, where Wm82.a6 matches the 'Williams' haplotype while the other two near-gapless assemblies do not match the haplotype of either parent of Williams 82. In addition to the Wm82.a6 assembly, we also assembled the genome of 'Fiskeby III,' a rich resource for abiotic stress resistance genes. A genome comparison of Wm82.a6 with Fiskeby III revealed the nucleotide and structural polymorphisms between the two genomes within a QTL region for iron deficiency chlorosis resistance. The Wm82.a6 and Fiskeby III genomes described here will enhance comparative and functional genomics capacities and applications in the soybean community.

The first two complete mitochondrial genomes for the genus Anagyrus (Hymenoptera, Encyrtidae) and their phylogenetic implications.

Anagyrus, a genus of Encyrtidae (Hymenoptera, Chalcidoidea), represents a successful group of parasitoid insects that attack various mealybug pests of agricultural and forestry plants. Until now, only 20 complete mitochondrial genomes have been sequenced, including those in this study. To enrich the diversity of mitochondrial genomes in Encyrtidae and to gain insights into their phylogenetic relationships, the mitochondrial genomes of two species of Anagyrus were sequenced, and the mitochondrial genomes of these species were compared and analyzed. Encyrtid mitochondrial genomes exhibit similarities in nucleotide composition, gene organization, and control region patterns. Comparative analysis of protein-coding genes revealed varying molecular evolutionary rates among different genes, with six genes (ATP8, ND2, ND4L, ND6, ND4 and ND5) showing higher rates than others. A phylogenetic analysis based on mitochondrial genome sequences supports the monophyly of Encyrtidae; however, the two subfamilies, Encyrtinae and Tetracneminae, are non-monophyletic. This study provides valuable insights into the phylogenetic relationships within the Encyrtidae and underscores the utility of mitochondrial genomes in the systematics of this family.

Reversible acetylation of HDAC8 regulates cell cycle.

HDAC8, a member of class I HDACs, plays a pivotal role in cell cycle regulation by deacetylating the cohesin subunit SMC3. While cyclins and CDKs are well-established cell cycle regulators, our knowledge of other regulators remains limited. Here we reveal the acetylation of K202 in HDAC8 as a key cell cycle regulator responsive to stress. K202 acetylation in HDAC8, primarily catalyzed by Tip60, restricts HDAC8 activity, leading to increased SMC3 acetylation and cell cycle arrest. Furthermore, cells expressing the mutant form of HDAC8 mimicking K202 acetylation display significant alterations in gene expression, potentially linked to changes in 3D genome structure, including enhanced chromatid loop interactions. K202 acetylation impairs cell cycle progression by disrupting the expression of cell cycle-related genes and sister chromatid cohesion, resulting in G2/M phase arrest. These findings indicate the reversible acetylation of HDAC8 as a cell cycle regulator, expanding our understanding of stress-responsive cell cycle dynamics.

The first complete chloroplast genome of Cyclamen persicum and its phylogenetic position within Primulaceae.

Cyclamen persicum Mill., 1768, is a perennial herb of Primulaceae and is native to the Mediterranean region. In this study, we sequenced the chloroplast genome of Cyclamen persicum. Its complete chloroplast contained 151,911 nucleotides, including a large single-copy (LSC) region with a length of 83,191 bp, a small single-copy (SSC) region with a length of 17,922 bp, and a pair of reverse repeat IR regions (25,399 bp). The C. persicum chloroplast genome encoded 112 unique genes, including 78 protein-coding genes, 30 tRNA genes, and four rRNA genes. The GC content of the entire genome was 37.25%, lower than that of many angiosperm plastome. The phylogenetic result indicated that C. persicum exhibited the closest relationship with Cyclamen rohlfsianum, and provided new information for the phylogeny relationship of genus Cyclamen.

The complete chloroplast genome sequence of Ampelopsis delavayana Planchon. ex Franch 1886 (Vitaceae).

Ampelopsis delavayana Planchon. ex Franch 1886 is a plant with significant pharmacological effects and ornamental importance. This research unveiled the complete chloroplast (cp) genome sequence of A. delavayana. The study highlights that the cp genome of A. delavayana possesses a distinct tetrameric structure spanning 162,497 base pairs, comprising a small single-copy (SSC) region of 18,902 base pairs, a large single-copy (LSC) region of 90,441 base pairs, and two inverted-repeat regions (IRs), each 26,577 base pairs in length. The GC content of the SSC, LSC, and IR regions of the genome was 31.80%, 35.16%, and 42.82%, respectively, culminating in an overall GC content of 37.27%. The genome comprised 130 genes, which included eight rRNAs, 36 tRNAs, and 86 protein-coding genes. Through phylogenetic analysis utilizing the maximum-likelihood method, it was established that A. delavayana was closely related to Ampelopsis glandulosa var. brevipedunculata, positioning it as a sister species. This report not only provides a scientific reference for understanding the phylogeny of the family Vitaceae but also enriches our genetic information of Ampelopsis.

A three-dimensional genomics view for speciation research.

Genomic information is folded in a three-dimensional (3D) structure, a rarely explored evolutionary driver of speciation. Technological advances now enable the study of 3D genome structures (3DGSs) across the Tree of Life. At the onset of 3D speciation genomics, we discuss the putative roles of 3DGSs in speciation.

Plastid DNA is a major source of nuclear genome complexity and of RNA genes in the orphan crop moringa.

BACKGROUND: Unlike Transposable Elements (TEs) and gene/genome duplication, the role of the so-called nuclear plastid DNA sequences (NUPTs) in shaping the evolution of genome architecture and function remains poorly studied. We investigate here the functional and evolutionary fate of NUPTs in the orphan crop Moringa oleifera (moringa), featured by the highest fraction of plastid DNA found so far in any plant genome, focusing on (i) any potential biases in their distribution in relation to specific nuclear genomic features, (ii) their contribution to the emergence of new genes and gene regions, and (iii) their impact on the expression of target nuclear genes. RESULTS: In agreement with their potential mutagenic effect, NUPTs are underrepresented among structural genes, although their overall transcription levels and broadness were only lower when involved exonic regions; the occurrence of plastid DNA generally did not result in a broader expression, except among those affected in introns by older NUPTs. In contrast, we found a strong enrichment of NUPTs among specific superfamilies of retrotransposons and several classes of RNA genes, including those participating in the protein biosynthetic machinery (i.e., rRNA and tRNA genes) and a specific class of regulatory RNAs. A significant fraction of NUPT RNA genes was found to be functionally expressed, thus potentially contributing to the nuclear pool. CONCLUSIONS: Our results complete our view of the molecular factors driving the evolution of nuclear genome architecture and function, and support plastid DNA in moringa as a major source of (i) genome complexity and (ii) the nuclear pool of RNA genes.

Dory: Computation of persistence diagrams up to dimension two for Vietoris-Rips filtrations of large data sets.

Persistent homology (PH) is an approach to topological data analysis (TDA) that computes multi-scale topologically invariant properties of high-dimensional data that are robust to noise. While PH has revealed useful patterns across various applications, computational requirements have limited applications to small data sets of a few thousand points. We present Dory, an efficient and scalable algorithm that can compute the persistent homology of sparse Vietoris-Rips complexes on larger data sets, up to and including dimension two and over the field Z2. As an application, we compute the PH of the human genome at high resolution as revealed by a genome-wide Hi-C data set containing approximately three million points. Extant algorithms were unable to process it, whereas Dory processed it within five minutes, using less than five GB of memory. Results show that the topology of the human genome changes significantly upon treatment with auxin, a molecule that degrades cohesin, corroborating the hypothesis that cohesin plays a crucial role in loop formation in DNA.

Single-cell 3D genome structure reveals distinct human pluripotent states.

BACKGROUND: Pluripotent states of embryonic stem cells (ESCs) with distinct transcriptional profiles affect ESC differentiative capacity and therapeutic potential. Although single-cell RNA sequencing has revealed additional subpopulations and specific features of naive and primed human pluripotent stem cells (hPSCs), the underlying mechanisms that regulate their specific transcription and that control their pluripotent states remain elusive. RESULTS: By single-cell analysis of high-resolution, three-dimensional (3D) genomic structure, we herein demonstrate that remodeling of genomic structure is highly associated with the pluripotent states of human ESCs (hESCs). The naive pluripotent state is featured with specialized 3D genomic structures and clear chromatin compartmentalization that is distinct from the primed state. The naive pluripotent state is achieved by remodeling the active euchromatin compartment and reducing chromatin interactions at the nuclear center. This unique genomic organization is linked to enhanced chromatin accessibility on enhancers and elevated expression levels of naive pluripotent genes localized to this region. In contradistinction, the primed state exhibits intermingled genomic organization. Moreover, active euchromatin and primed pluripotent genes are distributed at the nuclear periphery, while repressive heterochromatin is densely concentrated at the nuclear center, reducing chromatin accessibility and the transcription of naive genes. CONCLUSIONS: Our data provide insights into the chromatin structure of ESCs in their naive and primed states, and we identify specific patterns of modifications in transcription and chromatin structure that might explain the genes that are differentially expressed between naive and primed hESCs. Thus, the inversion or relocation of heterochromatin to euchromatin via compartmentalization is related to the regulation of chromatin accessibility, thereby defining pluripotent states and cellular identity.

Comparative 3D genome analysis between neural retina and retinal pigment epithelium reveals differential cis-regulatory interactions at retinal disease loci.

BACKGROUND: Vision depends on the interplay between photoreceptor cells of the neural retina and the underlying retinal pigment epithelium (RPE). Most genes involved in inherited retinal diseases display specific spatiotemporal expression within these interconnected retinal components through the local recruitment of cis-regulatory elements (CREs) in 3D nuclear space. RESULTS: To understand the role of differential chromatin architecture in establishing tissue-specific expression at inherited retinal disease loci, we mapped genome-wide chromatin interactions using in situ Hi-C and H3K4me3 HiChIP on neural retina and RPE/choroid from human adult donor eyes. We observed chromatin looping between active promoters and 32,425 and 8060 candidate CREs in the neural retina and RPE/choroid, respectively. A comparative 3D genome analysis between these two retinal tissues revealed that 56% of 290 known inherited retinal disease genes were marked by differential chromatin interactions. One of these was ABCA4, which is implicated in the most common autosomal recessive inherited retinal disease. We zoomed in on retina- and RPE-specific cis-regulatory interactions at the ABCA4 locus using high-resolution UMI-4C. Integration with bulk and single-cell epigenomic datasets and in vivo enhancer assays in zebrafish revealed tissue-specific CREs interacting with ABCA4. CONCLUSIONS: Through comparative 3D genome mapping, based on genome-wide, promoter-centric, and locus-specific assays of human neural retina and RPE, we have shown that gene regulation at key inherited retinal disease loci is likely mediated by tissue-specific chromatin interactions. These findings do not only provide insight into tissue-specific regulatory landscapes at retinal disease loci, but also delineate the search space for non-coding genomic variation underlying unsolved inherited retinal diseases.

Plastome structure, phylogeny and evolution of plastid genes in Reevesia (Helicteroideae, Malvaceae).

Reevesia is an eastern Asian-eastern North American disjunction genus in the family Malvaceae s.l. and comprises approximately 25 species. The relationships within the genus are not well understood. Here, 15 plastomes representing 12 Reevesia species were compared, with the aim of better understanding the species circumscription and phylogenetic relationships within the genus and among genera in the family Malvaceae s.l. The 11 newly sequenced plastomes range between 161,532 and 161, 945 bp in length. The genomes contain 114 unique genes, 18 of which are duplicated in the inverted repeats (IRs). Gene content of these plastomes is nearly identical. All the protein-coding genes are under purifying selection in the Reevesia plastomes compared. The top ten hypervariable regions, SSRs, and the long repeats identified are potential molecular markers for future population genetic and phylogenetic studies. Phylogenetic analysis based on the whole plastomes confirmed the monophyly of Reevesia and a close relationship with Durio (traditional Bombacaceae) in subfamily Helicteroideae, but not with the morphologically similar genera Pterospermum and Sterculia (both of traditional Sterculiaceae). Phylogenetic relationships within Reevesia suggested that two species, R. pubescens and R. thyrsoidea, as newly defined, are not monophyletic. Six taxa, R. membranacea, R. xuefengensis, R. botingensis, R. lofouensis, R. longipetiolata and R. pycnantha, are suggested to be recognized.

Consanguinty and its impact on health and genome dynamic: An example from Tunisia.

The genetic disease spectrum in Tunisia arises from the founder effect, genetic drift, selection, and consanguinity. The latter represents a deviation from panmixia, characterized by a non-random matrimonial choice that may be subject to several rules, such as socio-cultural, economic, or other factors. This shifts the genetic structure away from the Hardy-Weinberg equilibrium, increasing homozygous genotypes and decreasing heterozygotes, thus raising the frequency of autosomal recessive diseases. Similar to other Arab populations, Tunisia displays high consanguinity rates that vary geographically. Approximately 60% of reported diseases in Tunisia are autosomal recessive, with consanguinity possibly occurring in 80% of families for a specific disease. In inbred populations, consanguinity amplifies autosomal recessive disease risk, yet it does not influence autosomal dominant disease likelihood but rather impacts its phenotype. Consanguinity is also suggested to be a major factor in the homozygosity of deleterious variants leading to comorbid expression. At the genome level, inbred individuals inherit homozygous mutations and adjacent genomic regions known as runs of homozygosity (ROHs). Short ROHs indicate distant inbreeding, while long ROHs refer to recent inbreeding. ROHs are distributed rather irregularly across the genome, with certain short regions featuring an excess of ROH, known as ROH islands. In this review, we discuss consanguinity's impact on population health and genome dynamics, using Tunisia as a model.

Modulated illumination microscopy: Application perspectives in nuclear nanostructure analysis.

The structure of the cell nucleus of higher organisms has become a major topic of advanced light microscopy. So far, a variety of methods have been applied, including confocal laser scanning fluorescence microscopy, 4Pi, STED and localisation microscopy approaches, as well as different types of patterned illumination microscopy, modulated either laterally (in the object plane) or axially (along the optical axis). Based on our experience, we discuss here some application perspectives of Modulated Illumination Microscopy (MIM) and its combination with single-molecule localisation microscopy (SMLM). For example, spatially modulated illumination microscopy/SMI (illumination modulation along the optical axis) has been used to determine the axial extension (size) of small, optically isolated fluorescent objects between ≤ 200 nm and ≥ 40 nm diameter with a precision down to the few nm range; it also allows the axial positioning of such structures down to the 1 nm scale; combined with laterally structured illumination/SIM, a 3D localisation precision of ≤1 nm is expected using fluorescence yields typical for SMLM applications. Together with the nanosizing capability of SMI, this can be used to analyse macromolecular nuclear complexes with a resolution approaching that of cryoelectron microscopy.

Genome and evolution of Tibet orbivirus, TIBOV (genus Orbivirus, family Reoviridae).

Tibet orbivirus (TIBOV) was first isolated from Anopheles maculatus mosquitoes in Xizang, China, in 2009. In recent years, more TIBOV strains have been isolated in several provinces across China, Japan, East Asia, and Nepal, South Asia. Furthermore, TIBOVs have also been isolated from Culex mosquitoes, and several midge species. Additionally, TIBOV neutralizing antibodies have been detected in serum specimens from several mammals, including cattle, sheep, and pigs. All of the evidence suggests that the geographical distribution of TIBOVs has significantly expanded in recent years, with an increased number of vector species involved in its transmission. Moreover, the virus demonstrated infectivity towards a variety of animals. Although TIBOV is considered an emerging orbivirus, detailed reports on its genome and molecular evolution are currently lacking. Thus, this study performed the whole-genome nucleotide sequencing of three TIBOV isolates from mosquitoes and midges collected in China in 2009, 2011, and 2019. Furthermore, the genome and molecular genetic evolution of TIBOVs isolated from different countries, periods, and hosts (mosquitoes, midges, and cattle) was systematically analyzed. The results revealed no molecular specificity among TIBOVs isolated from different countries, periods, and vectors. Meanwhile, the time-scaled phylogenetic analysis demonstrated that the most recent common ancestor (TMRCA) of TIBOV appeared approximately 797 years ago (95% HPD: 16-2347) and subsequently differentiated at least three times, resulting in three distinct genotypes. The evolutionary rate of TIBOVs was about 2.12 × 10-3 nucleotide substitutions per site per year (s/s/y) (95% HPD: 3.07 × 10-5, 9.63 × 10-3), which is similar to that of the bluetongue virus (BTV), also in the Orbivirus genus. Structural analyses of the viral proteins revealed that the three-dimensional structures of the outer capsid proteins of TIBOV and BTV were similar. These results suggest that TIBOV is a newly discovered and rapidly evolving virus transmitted by various blood-sucking insects. Given the potential public health burden of this virus and its high infectious rate in a wide range of animals, it is significant to strengthen research on the genetic variation of TIBOVs in blood-feeding insects and mammals in the natural environment and the infection status in animals.

Enhancer-promoter interactions are reconfigured through the formation of long-range multiway hubs as mouse ES cells exit pluripotency.

Enhancers bind transcription factors, chromatin regulators, and non-coding transcripts to modulate the expression of target genes. Here, we report 3D genome structures of single mouse ES cells as they are induced to exit pluripotency and transition through a formative stage prior to undergoing neuroectodermal differentiation. We find that there is a remarkable reorganization of 3D genome structure where inter-chromosomal intermingling increases dramatically in the formative state. This intermingling is associated with the formation of a large number of multiway hubs that bring together enhancers and promoters with similar chromatin states from typically 5-8 distant chromosomal sites that are often separated by many Mb from each other. In the formative state, genes important for pluripotency exit establish contacts with emerging enhancers within these multiway hubs, suggesting that the structural changes we have observed may play an important role in modulating transcription and establishing new cell identities.

Host-specific co-evolution likely driven by diet in Buchnera aphidicola.

BACKGROUND: Russian wheat aphid (Diuraphis noxia Kurd.) is a severe pest to wheat, and even though resistance varieties are available to curb this pest, they are becoming obsolete with the development of new virulent aphid populations. Unlike many other aphids, D noxia only harbours a single endosymbiont, Buchnera aphidicola. Considering the importance of Buchnera, this study aimed to elucidate commonalities and dissimilarities between various hosts, to better understand its distinctiveness within its symbiotic relationship with D. noxia. To do so, the genome of the D. noxia's Buchnera was assembled and compared to those of other aphid species that feed on diverse host species. RESULTS: The overall importance of several features such as gene length and percentage GC content was found to be critical for the maintenance of Buchnera genes when compared to their closest free-living relative, Escherichia coli. Buchnera protein coding genes were found to have percentage GC contents that tended towards a mean of ~ 26% which had strong correlation to their identity to their E. coli homologs. Several SNPs were identified between different aphid populations and multiple isolates of Buchnera were confirmed in single aphids. CONCLUSIONS: Establishing the strong correlation of percentage GC content of protein coding genes and gene identity will allow for identifying which genes will be lost in the continually shrinking Buchnera genome. This is also the first report of a parthenogenically reproducing aphid that hosts multiple Buchnera strains in a single aphid, raising questions regarding the benefits of maintaining multiple strains. We also found preliminary evidence for post-transcriptional regulation of Buchnera genes in the form of polyadenylation.