Proximity Ligation Data Research Articles

Chromosome-Level Genome Assembly and Annotation of a Periodical Cicada Species: Magicicada septendecula.

We present a high-quality assembly and annotation of the periodical cicada species, Magicicada septendecula (Hemiptera: Auchenorrhyncha: Cicadidae). Periodical cicadas have a significant ecological impact, serving as a food source for many mammals, reptiles, and birds. Magicicada are well known for their massive emergences of 1 to 3 species that appear in different locations in the eastern United States nearly every year. These year classes ("broods") emerge dependably every 13 or 17 yr in a given location. Recently, it has become clear that 4-yr early or late emergences of a sizeable portion of a population are an important part of the history of brood formation; however, the biological mechanisms by which they track the passage of time remain a mystery. Using PacBio HiFi reads in conjunction with Hi-C proximity ligation data, we have assembled and annotated the first whole genome for a periodical cicada, an important resource for future phylogenetic and comparative genomic analysis. This also represents the first quality genome assembly and annotation for the Hemipteran superfamily Cicadoidea. With a scaffold N50 of 518.9 Mb and a complete BUSCO score of 96.7%, we are confident that this assembly will serve as a vital resource toward uncovering the genomic basis of periodical cicadas' long, synchronized life cycles and will provide a robust framework for further investigations into these insects.

A computationally-enhanced hiCLIP atlas reveals Staufen1-RNA binding features and links 3' UTR structure to RNA metabolism.

The structure of mRNA molecules plays an important role in its interactions with trans-acting factors, notably RNA binding proteins (RBPs), thus contributing to the functional consequences of this interplay. However, current transcriptome-wide experimental methods to chart these interactions are limited by their poor sensitivity. Here we extend the hiCLIP atlas of duplexes bound by Staufen1 (STAU1) ∼10-fold, through careful consideration of experimental assumptions, and the development of bespoke computational methods which we apply to existing data. We present Tosca, a Nextflow computational pipeline for the processing, analysis and visualisation of proximity ligation sequencing data generally. We use our extended duplex atlas to discover insights into the RNA selectivity of STAU1, revealing the importance of structural symmetry and duplex-span-dependent nucleotide composition. Furthermore, we identify heterogeneity in the relationship between transcripts with STAU1-bound 3' UTR duplexes and metabolism of the associated RNAs that we relate to RNA structure: transcripts with short-range proximal 3' UTR duplexes have high degradation rates, but those with long-range duplexes have low rates. Overall, our work enables the integrative analysis of proximity ligation data delivering insights into specific features and effects of RBP-RNA structure interactions.

Genome assembly of the deep-sea coral Lophelia pertusa.

Like their shallow-water counterparts, cold-water corals create reefs that support highly diverse communities, and these structures are subject to numerous anthropogenic threats. Here, we present the genome assembly of Lophelia pertusa from the southeastern coast of the USA, the first one for a deep-sea scleractinian coral species. We generated PacBio continuous long reads data for an initial assembly and proximity ligation data for scaffolding. The assembly was annotated using evidence from transcripts, proteins, and ab initio gene model predictions. This assembly is comparable to high-quality reference genomes from shallow-water scleractinian corals. The assembly comprises 2,858 scaffolds (N50 1.6Mbp) and has a size of 556.9Mbp. Approximately 57% of the genome comprises repetitive elements and 34% of coding DNA. We predicted 41,089 genes, including 91.1% of complete metazoan orthologs. This assembly will facilitate investigations into the ecology of this species and the evolution of deep-sea corals.

A chromosome-scale high-contiguity genome assembly of the cheetah (Acinonyx jubatus).

The cheetah (Acinonyx jubatus, SCHREBER 1775) is a large felid and is considered the fastest land animal. Historically, it inhabited open grassland across Africa, the Arabian Peninsula, and southwestern Asia; however, only small and fragmented populations remain today. Here, we present a de novo genome assembly of the cheetah based on PacBio continuous long reads and Hi-C proximity ligation data. The final assembly (VMU_Ajub_asm_v1.0) has a total length of 2.38 Gb, of which 99.7% are anchored into the expected 19 chromosome-scale scaffolds. The contig and scaffold N50 values of 96.8 Mb and 144.4 Mb, respectively, a BUSCO completeness of 95.4% and a k-mer completeness of 98.4%, emphasize the high quality of the assembly. Furthermore, annotation of the assembly identified 23,622 genes and a repeat content of 40.4%. This new highly contiguous and chromosome-scale assembly will greatly benefit conservation and evolutionary genomic analyses and will be a valuable resource, e.g., to gain a detailed understanding of the function and diversity of immune response genes in felids.

Genome report: chromosome-level draft assemblies of the snow leopard, African leopard, and tiger (Panthera uncia, Panthera pardus pardus, and Panthera tigris).

The big cats (genus Panthera) represent some of the most popular and charismatic species on the planet. Although some reference genomes are available for this clade, few are at the chromosome level, inhibiting high-resolution genomic studies. We assembled genomes from 3 members of the genus, the tiger (Panthera tigris), the snow leopard (Panthera uncia), and the African leopard (Panthera pardus pardus), at chromosome or near-chromosome level. We used a combination of short- and long-read technologies, as well as proximity ligation data from Hi-C technology, to achieve high continuity and contiguity for each individual. We hope that these genomes will aid in further evolutionary and conservation research of this iconic group of mammals.

Whole-genome sequence and assembly of the Javan gibbon (Hylobates moloch).

The Javan gibbon, Hylobates moloch, is an endangered gibbon species restricted to the forest remnants of western and central Java, Indonesia, and one of the rarest of the Hylobatidae family. Hylobatids consist of 4 genera (Holoock, Hylobates, Symphalangus, and Nomascus) that are characterized by different numbers of chromosomes, ranging from 38 to 52. The underlying cause of this karyotype plasticity is not entirely understood, at least in part, due to the limited availability of genomic data. Here we present the first scaffold-level assembly for H. moloch using a combination of whole-genome Illumina short reads, 10X Chromium linked reads, PacBio, and Oxford Nanopore long reads and proximity-ligation data. This Hylobates genome represents a valuable new resource for comparative genomics studies in primates.

A haploid pseudo-chromosome genome assembly for a keystone sagebrush species of western North American rangelands.

Increased ecological disturbances, species invasions, and climate change are creating severe conservation problems for several plant species that are widespread and foundational. Understanding the genetic diversity of these species and how it relates to adaptation to these stressors are necessary for guiding conservation and restoration efforts. This need is particularly acute for big sagebrush (Artemisia tridentata; Asteraceae), which was once the dominant shrub over 1,000,000 km2 in western North America but has since retracted by half and thus has become the target of one of the largest restoration seeding efforts globally. Here, we present the first reference-quality genome assembly for an ecologically important subspecies of big sagebrush (A. tridentata subsp. tridentata) based on short and long reads, as well as chromatin proximity ligation data analyzed using the HiRise pipeline. The final 4.2-Gb assembly consists of 5,492 scaffolds, with nine pseudo-chromosomal scaffolds (nine scaffolds comprising at least 90% of the assembled genome; n = 9). The assembly contains an estimated 43,377 genes based on ab initio gene discovery and transcriptional data analyzed using the MAKER pipeline, with 91.37% of BUSCOs being completely assembled. The final assembly was highly repetitive, with repeat elements comprising 77.99% of the genome, making the Artemisia tridentata subsp. tridentata genome one of the most highly repetitive plant genomes to be sequenced and assembled. This genome assembly advances studies on plant adaptation to drought and heat stress and provides a valuable tool for future genomic research.

Developmental pyrethroid exposure and age influence phenotypes in a Chd8 haploinsufficient autism mouse model

Hundreds of genes have been associated with autism spectrum disorder (ASD), including loss-of-function mutations in chromodomain helicase DNA binding protein 8 (Chd8). Environmental factors also are implicated in autism risk and have the potential to exacerbate phenotypes in genetically sensitized backgrounds. Here we investigate transcriptional and behavioral phenotypes in a Chd8 haploinsufficient (Chd8V986*/+) mouse line exposed to the pesticide deltamethrin (DM) from conception to postnatal day 22. Vehicle-exposed Chd8V986*/+ mice displayed ASD-associated phenotypes, including anxiety-like behavior and altered sociability, replicating a previous study with this mouse line. A core set of genes was altered in Chd8V986*/+ mice at multiple ages, including Usp11, Wars2, Crlf2, and Eglf6, and proximity ligation data indicated direct binding of CHD8 to the 5’ region of these genes. Moreover, oligodendrocyte and neurodegenerative transcriptional phenotypes were apparent in 12 and 18 month old Chd8V986*/+ mice. Following DM exposure, the mutant mice displayed an exacerbated phenotype in the elevated plus maze, and genes associated with vascular endothelial cells were downregulated in the cerebral cortex of older Chd8V986*/+ animals. Our study reveals a gene x environment interaction with a Chd8 haploinsufficient mouse line and points to the importance of investigating phenotypes in ASD animal models across the lifespan.

Global mapping of RNA homodimers in living cells.

RNA homodimerization is important for various physiological processes, including the assembly of membraneless organelles, RNA subcellular localization, and packaging of viral genomes. However, understanding RNA dimerization has been hampered by the lack of systematic in vivo detection methods. Here, we show that CLASH, PARIS, and other RNA proximity ligation methods detect RNA homodimers transcriptome-wide as “overlapping” chimeric reads that contain more than one copy of the same sequence. Analyzing published proximity ligation data sets, we show that RNA:RNA homodimers mediated by direct base-pairing are rare across the human transcriptome, but highly enriched in specific transcripts, including U8 snoRNA, U2 snRNA, and a subset of tRNAs. Mutations in the homodimerization domain of U8 snoRNA impede dimerization in vitro and disrupt zebrafish development in vivo, suggesting an evolutionarily conserved role of this domain. Analysis of virus-infected cells reveals homodimerization of SARS-CoV-2 and Zika genomes, mediated by specific palindromic sequences located within protein-coding regions of N gene in SARS-CoV-2 and NS2A gene in Zika. We speculate that regions of viral genomes involved in homodimerization may constitute effective targets for antiviral therapies.

Novel canine high-quality metagenome-assembled genomes, prophages and host-associated plasmids provided by long-read metagenomics together with Hi-C proximity ligation.

The human gut microbiome has been extensively studied, yet the canine gut microbiome is still largely unknown. The availability of high-quality genomes is essential in the fields of veterinary medicine and nutrition to unravel the biological role of key microbial members in the canine gut environment. Our aim was to evaluate nanopore long-read metagenomics and Hi-C (high-throughput chromosome conformation capture) proximity ligation to provide high-quality metagenome-assembled genomes (HQ MAGs) of the canine gut environment. By combining nanopore long-read metagenomics and Hi-C proximity ligation, we retrieved 27 HQ MAGs and 7 medium-quality MAGs of a faecal sample of a healthy dog. Canine MAGs (CanMAGs) improved genome contiguity of representatives from the animal and human MAG catalogues – short-read MAGs from public datasets – for the species they represented: they were more contiguous with complete ribosomal operons and at least 18 canonical tRNAs. Both canine-specific bacterial species and gut generalists inhabit the dog’s gastrointestinal environment. Most of them belonged to Firmicutes , followed by Bacteroidota and Proteobacteria . We also assembled one Actinobacteriota and one Fusobacteriota MAG. CanMAGs harboured antimicrobial-resistance genes (ARGs) and prophages and were linked to plasmids. ARGs conferring resistance to tetracycline were most predominant within CanMAGs, followed by lincosamide and macrolide ones. At the functional level, carbohydrate transport and metabolism was the most variable within the CanMAGs, and mobilome function was abundant in some MAGs. Specifically, we assigned the mobilome functions and the associated mobile genetic elements to the bacterial host. The CanMAGs harboured 50 bacteriophages, providing novel bacterial-host information for eight viral clusters, and Hi-C proximity ligation data linked the six potential plasmids to their bacterial host. Long-read metagenomics and Hi-C proximity ligation are likely to become a comprehensive approach to HQ MAG discovery and assignment of extra-chromosomal elements to their bacterial host. This will provide essential information for studying the canine gut microbiome in veterinary medicine and animal nutrition.

Assembled and annotated 26.5 Gbp coast redwood genome: a resource for estimating evolutionary adaptive potential and investigating hexaploid origin

Sequencing, assembly, and annotation of the 26.5 Gbp hexaploid genome of coast redwood (Sequoia sempervirens) was completed leading toward discovery of genes related to climate adaptation and investigation of the origin of the hexaploid genome. Deep-coverage short-read Illumina sequencing data from haploid tissue from a single seed were combined with long-read Oxford Nanopore Technologies sequencing data from diploid needle tissue to create an initial assembly, which was then scaffolded using proximity ligation data to produce a highly contiguous final assembly, SESE 2.1, with a scaffold N50 size of 44.9 Mbp. The assembly included several scaffolds that span entire chromosome arms, confirmed by the presence of telomere and centromere sequences on the ends of the scaffolds. The structural annotation produced 118,906 genes with 113 containing introns that exceed 500 Kbp in length and one reaching 2 Mb. Nearly 19 Gbp of the genome represented repetitive content with the vast majority characterized as long terminal repeats, with a 2.9:1 ratio of Copia to Gypsy elements that may aid in gene expression control. Comparison of coast redwood to other conifers revealed species-specific expansions for a plethora of abiotic and biotic stress response genes, including those involved in fungal disease resistance, detoxification, and physical injury/structural remodeling and others supporting flavonoid biosynthesis. Analysis of multiple genes that exist in triplicate in coast redwood but only once in its diploid relative, giant sequoia, supports a previous hypothesis that the hexaploidy is the result of autopolyploidy rather than any hybridizations with separate but closely related conifer species.

Hi-CO: 3D genome structure analysis with nucleosome resolution.

The nucleosome is the basic organizational unit of the genome. The folding structure of nucleosomes is closely related to genome functions, and has been reported to be in dynamic interplay with binding of various nuclear proteins to genomic loci. Here, we describe our high-throughput chromosome conformation capture with nucleosome orientation (Hi-CO) technology to derive 3D nucleosome positions with their orientations at every genomic locus in the nucleus. This technology consists of an experimental procedure for nucleosome proximity analysis and a computational procedure for 3D modeling. The experimental procedure is based on an improved method of high-throughput chromosome conformation capture (Hi-C) analysis. Whereas conventional Hi-C allows spatial proximity analysis among genomic loci with 1-10 kbp resolution, our Hi-CO allows proximity analysis among DNA entry or exit points at every nucleosome locus. This analysis is realized by carrying out ligations among the entry/exit points in every nucleosome in a micrococcal-nuclease-fragmented genome, and by quantifying frequencies of ligation products with next-generation sequencing. Our protocol has enabled this analysis by cleanly excluding unwanted non-ligation products that are abundant owing to the frequent genome fragmentation by micrococcal nuclease. The computational procedure is based on simulated annealing-molecular dynamics, which allows determination of optimized 3D positions and orientations of every nucleosome that satisfies the proximity ligation data sufficiently well. Typically, examination of the Saccharomyces cerevisiae genome with 130 million sequencing reads facilitates analysis of a total of 66,360 nucleosome loci with 6.8 nm resolution. The technique requires 2-3 weeks for sequencing library preparation and 2 weeks for simulation.

Chromosome-Level Assembly of the Atlantic Silverside Genome Reveals Extreme Levels of Sequence Diversity and Structural Genetic Variation.

The levels and distribution of standing genetic variation in a genome can provide a wealth of insights about the adaptive potential, demographic history, and genome structure of a population or species. As structural variants are increasingly associated with traits important for adaptation and speciation, investigating both sequence and structural variation is essential for wholly tapping this potential. Using a combination of shotgun sequencing, 10x Genomics linked reads and proximity-ligation data (Chicago and Hi-C), we produced and annotated a chromosome-level genome assembly for the Atlantic silverside (Menidia menidia)—an established ecological model for studying the phenotypic effects of natural and artificial selection—and examined patterns of genomic variation across two individuals sampled from different populations with divergent local adaptations. Levels of diversity varied substantially across each chromosome, consistently being highly elevated near the ends (presumably near telomeric regions) and dipping to near zero around putative centromeres. Overall, our estimate of the genome-wide average heterozygosity in the Atlantic silverside is among the highest reported for a fish, or any vertebrate (1.32–1.76% depending on inference method and sample). Furthermore, we also found extreme levels of structural variation, affecting ∼23% of the total genome sequence, including multiple large inversions (> 1 Mb and up to 12.6 Mb) associated with previously identified haploblocks showing strong differentiation between locally adapted populations. These extreme levels of standing genetic variation are likely associated with large effective population sizes and may help explain the remarkable adaptive divergence among populations of the Atlantic silverside.

The genome of Draba nivalis shows signatures of adaptation to the extreme environmental stresses of the Arctic.

The Arctic is one of the most extreme terrestrial environments on the planet. Here, we present the first chromosome‐scale genome assembly of a plant adapted to the high Arctic, Draba nivalis (Brassicaceae), an attractive model species for studying plant adaptation to the stresses imposed by this harsh environment. We used an iterative scaffolding strategy with data from short‐reads, single‐molecule long reads, proximity ligation data, and a genetic map to produce a 302 Mb assembly that is highly contiguous with 91.6% assembled into eight chromosomes (the base chromosome number). To identify candidate genes and gene families that may have facilitated adaptation to Arctic environmental stresses, we performed comparative genomic analyses with nine non‐Arctic Brassicaceae species. We show that the D. nivalis genome contains expanded suites of genes associated with drought and cold stress (e.g., related to the maintenance of oxidation‐reduction homeostasis, meiosis, and signaling pathways). The expansions of gene families associated with these functions appear to be driven in part by the activity of transposable elements. Tests of positive selection identify suites of candidate genes associated with meiosis and photoperiodism, as well as cold, drought, and oxidative stress responses. Our results reveal a multifaceted landscape of stress adaptation in the D. nivalis genome, offering avenues for the continued development of this species as an Arctic model plant.

The Nucleome Data Bank: web-based resources to simulate and analyze the three-dimensional genome.

We introduce the Nucleome Data Bank (NDB), a web-based platform to simulate and analyze the three-dimensional (3D) organization of genomes. The NDB enables physics-based simulation of chromosomal structural dynamics through the MEGABASE + MiChroM computational pipeline. The input of the pipeline consists of epigenetic information sourced from the Encode database; the output consists of the trajectories of chromosomal motions that accurately predict Hi-C and fluorescence insitu hybridization data, as well as multiple observations of chromosomal dynamics in vivo. As an intermediate step, users can also generate chromosomal sub-compartment annotations directly from the same epigenetic input, without the use of any DNA–DNA proximity ligation data. Additionally, the NDB freely hosts both experimental and computational structural genomics data. Besides being able to perform their own genome simulations and download the hosted data, users can also analyze and visualize the same data through custom-designed web-based tools. In particular, the one-dimensional genetic and epigenetic data can be overlaid onto accurate 3D structures of chromosomes, to study the spatial distribution of genetic and epigenetic features. The NDB aims to be a shared resource to biologists, biophysicists and all genome scientists. The NDB is available at https://ndb.rice.edu.

Improved reference genome for the domestic horse increases assembly contiguity and composition

Recent advances in genomic sequencing technology and computational assembly methods have allowed scientists to improve reference genome assemblies in terms of contiguity and composition. EquCab2, a reference genome for the domestic horse, was released in 2007. Although of equal or better quality compared to other first-generation Sanger assemblies, it had many of the shortcomings common to them. In 2014, the equine genomics research community began a project to improve the reference sequence for the horse, building upon the solid foundation of EquCab2 and incorporating new short-read data, long-read data, and proximity ligation data. Here, we present EquCab3. The count of non-N bases in the incorporated chromosomes is improved from 2.33 Gb in EquCab2 to 2.41 Gb in EquCab3. Contiguity has also been improved nearly 40-fold with a contig N50 of 4.5 Mb and scaffold contiguity enhanced to where all but one of the 32 chromosomes is comprised of a single scaffold.

Higher-Order Organization Principles of Pre-translational mRNPs

Compared to noncoding RNAs (ncRNAs), such as rRNAs and ribozymes, for which high-resolution structures abound, little is known about the tertiary structures of mRNAs. In eukaryotic cells, newly made mRNAs are packaged with proteins in highlycompacted mRNA particles (mRNPs), but the manner of this mRNA compaction is unknown. Here, we developed and implemented RIPPLiT (RNA immunoprecipitation and proximity ligation in tandem), a transcriptome-wide method for probing the 3D conformations of RNAs stably associated with defined proteins, in this case, exon junction complex (EJC) core factors. EJCs multimerize with other mRNP components to form megadalton-sized complexes that protect large swaths of newly synthesized mRNAs from endonuclease digestion. Unlike ncRNPs, wherein strong locus-specific structures predominate, mRNPs behave more like flexible polymers. Polymer analysis of proximity ligation data for hundreds of mRNA species demonstrates that nascent and pre-translational mammalian mRNAs are compacted by their associated proteins into linear rod-like structures.

Genome assembly with in vitro proximity ligation data and whole-genome triplication in lettuce

Lettuce (Lactuca sativa) is a major crop and a member of the large, highly successful Compositae family of flowering plants. Here we present a reference assembly for the species and family. This was generated using whole-genome shotgun Illumina reads plus in vitro proximity ligation data to create large superscaffolds; it was validated genetically and superscaffolds were oriented in genetic bins ordered along nine chromosomal pseudomolecules. We identify several genomic features that may have contributed to the success of the family, including genes encoding Cycloidea-like transcription factors, kinases, enzymes involved in rubber biosynthesis and disease resistance proteins that are expanded in the genome. We characterize 21 novel microRNAs, one of which may trigger phasiRNAs from numerous kinase transcripts. We provide evidence for a whole-genome triplication event specific but basal to the Compositae. We detect 26% of the genome in triplicated regions containing 30% of all genes that are enriched for regulatory sequences and depleted for genes involved in defence.