Large Genome Research Articles

The expanded genome of Hexamita inflata, a free-living diplomonad

Diplomonads are anaerobic, flagellated protists, being part of the Metamonada group of Eukaryotes. Diplomonads either live as endobionts (parasites and commensals) of animals or free-living in low-oxygen environments. Genomic information is available for parasitic diplomonads like Giardia intestinalis and Spironucleus salmonicida, while little is known about the genomic arrangements of free-living diplomonads. We have generated the first reference genome of a free-living diplomonad, Hexamita inflata. The final version of the genome assembly is fragmented (1241 contigs) but substantially larger (142 Mbp) than the parasitic diplomonad genomes (9.8–14.7 Mbp). It encodes 79,341 proteins; 29,874 have functional annotations and 49,467 are hypothetical proteins. Interspersed repeats comprise 34% of the genome (9617 Retroelements, 2676 DNA transposons). The large expansion of protein-encoding capacity and the interspersed repeats are the major reasons for the large genome size. This genome from a free-living diplomonad will be the basis for further studies of the Diplomonadida lineage and the evolution of parasitism-free living style transitions.

K-mer approaches for biodiversity genomics.

The wide array of currently available genomes displays a wonderful diversity in size, composition, and structure and is quickly expanding thanks to several global biodiversity genomics initiatives. However, sequencing of genomes, even with the latest technologies, can still be challenging for both technical (e.g., small physical size, contaminated samples, or access to appropriate sequencing platforms) and biological reasons (e.g., germline-restricted DNA, variable ploidy levels, sex chromosomes, or very large genomes). In recent years, k-mer-based techniques have become popular to overcome some of these challenges. They are based on the simple process of dividing the analyzed sequences (e.g., raw reads or genomes) into a set of subsequences of length k, called k-mers, and then analyzing the frequency or sequences of those k-mers. Analyses based on k-mers allow for a rapid and intuitive assessment of complex sequencing data sets. Here, we provide a comprehensive review to the theoretical properties and practical applications of k-mers in biodiversity genomics with a special focus on genome modeling.

Pseudomonas aeruginosa maintains an inducible array of novel and diverse prophages over lengthy persistence in cystic fibrosis lungs.

Pseudomonas aeruginosa has increasing clinical relevance and commonly occupies the cystic fibrosis (CF) airways. Its ability to colonize and persist in diverse niches is attributed to its large accessory genome, where prophages represent a common feature and may contribute to its fitness and persistence. We focused on the CF airways niche and used 197 longitudinal isolates from 12 patients persistently infected by P. aeruginosa. We computationally predicted intact prophages for each longitudinal group and scored their long-term persistence. We then confirmed prophage inducibility and mapped their location in the host chromosome with lysate sequencing. Using comparative genomics, we evaluated prophage genomic diversity, long-term persistence and level of genomic maintenance. Our findings support previous findings that most P. aeruginosa genomes harbour prophages some of which can self-induce, and that a common CF-treating antibiotic, ciprofloxacin, can induce prophages. Induced prophage genomes displayed high diversity and even genomic novelty. Finally, all induced prophages persisted long-term with their genomes avoiding gene loss and degradation over four years of host replication in the stressful CF airways niche. This and our detection of phage genes which contribute to host competitiveness and adaptation, lends support to our hypothesis that the vast majority of prophages detected as intact and inducible in this study facilitated their host fitness and persistence.

Genome recombination on demand.

Large genome rearrangements in mammalian cells can be generated at scale.

Influence of Ty3/gypsy and Ty1/copia LTR-retrotransposons on the large genomes of Alstroemeriaceae: genome landscape of Bomarea edulis (Tussac) Herb.

Repetitive elements are the main components of many plant genomes and play a crucial role in the variation of genome size and structure, ultimately impacting species diversification and adaptation. Alstroemeriaceae exhibits species with large genomes, not attributed to polyploidy. In this study, we analysed the repetitive fraction of the genome of Bomarea edulis through low-coverage sequencing and in silico characterization, and compared it to the repeats of Alstroemeria longistaminea, a species from a sister genus that has been previously characterized. LTR-retrotransposons were identified as the most abundant elementsin the B. edulis genome (50.22%), with significant variations in abundance for specific lineages between the two species. The expansion of the B. edulis genome was likely due to three main lineages of LTR retrotransposons, Ty3/gypsy Tekay and Retand and Ty1/copia SIRE, all represented by truncated elements which were probably active in the past. Furthermore, the proportion of satDNA (~ 7%) was six times higher in B. edulis compared to A. longistaminea, with most families exhibiting a dispersed, uniform distribution in the genome. SatDNAs, thus, contributed to some extent to genome obesity. Despite diverging around 29 Mya, both species still share some satDNA families and retrotransposons. However, differences in repeat abundances and sequence variants led to genome differentiation despite their similar sizes and structure.

Transposon proliferation drives genome architecture and regulatory evolution in wild and domesticated peppers.

Pepper (Capsicum spp.) is a widely consumed vegetable with exceptionally large genomes in Solanaceae, yet its genomic evolutionary history remains largely unknown. Here we present 11 high-quality Capsicum genome assemblies, including two gap-free genomes, covering four wild and all five domesticated pepper species. We reconstructed the ancestral karyotype and inferred the evolutionary trajectory of peppers. The expanded and variable genome sizes were attributed to differential transposable element accumulations, which shaped 3D chromatin architecture and introduced mutations associated with traits such as fruit orientation and colour. Using a chromatin accessibility atlas of Capsicum, we highlight the influence of transposable elements on regulatory element evolution. Furthermore, by constructing a haploblock map of 124 pepper core germplasms, we uncover frequent introgressions that facilitate the formation of sweet blocky pepper and the acquisition of important traits such as resistance to pepper mild mottle virus. These findings on the genomic and functional evolution of Capsicum will benefit pepper breeding.

Distinct patterns of genome size evolution in each bryophyte lineage are not correlated with whole genome duplication.

Genome size varies by orders of magnitude across land plants, and the factors driving evolutionary increases and decreases in genome size vary across lineages. Bryophytes have the smallest genomes relative to other land plants and there is growing evidence for frequent whole genome duplication (WGD) across the lineage. However, the broad patterns of genome size, chromosome number, and WGD have yet to be characterized across bryophytes in a phylogenetic context. In the present study, we use a phylogenetic comparative approach and leverage previously published data on genome size, chromosome number, and WGD to reconstruct the evolutionary history of these traits across the three major bryophyte lineages: hornworts, liverworts, and mosses. We infer ancestral haploid chromosome numbers for each lineage and introduce a novel metric for assessing polyploidy using chromosome counts. Each lineage of bryophytes exhibits a distinct pattern of genome size evolution and prevalence of WGD, with mosses having the most dynamic genome sizes and highest propensity for WGD. We found that 21.3% of mosses and 13% of liverworts species have naturally occurring polyploids. In addition, haploid genome size (1C) is most dynamic in the mosses which includes 15 transitions to larger genomes and nine reversals, largely in the orders Dicranales and Hypnales. There is no correlation between genome size and WGD or genome size and chromosome number, potentially suggesting rapid genome downsizing following WGD. As bryophytes are poikilohydric (desiccation tolerant) plants, having large genomes may be physiologically prohibitive given the cost to growth and metabolism associated with them. These findings emphasize the unique evolution of the bryophytes broadly and of the hornworts, liverworts, and mosses individually, and should therefore serve as impetus for more in-depth experimental studies of genome size evolution and WGD in bryophytes.

A deep dive into bryophyte genome space reveals opposing evolutionary trends in the sister lineages, mosses and liverworts

AbstractAmong the lineages of the tree of life, land plants exhibit a remarkably high genomic disparity because of their distinct evolutionary trajectories in the phylogenetic history of their major lineages. The macroevolutionary pattern of genomic evolution has been mainly investigated to obtain insights into well‐studied lineages such as angiosperms, but little attention has been given to many important lineages such as bryophytes. This study was designed to resolve this gap by comparing the genomic evolution trajectories of mosses and liverworts. Thus, a data set comprising chromosome number and genome size was compiled, including previously published and newly generated data that were used to trace the phylogenetic history of these two parameters among mosses and liverworts via ancestral state reconstruction and phylogenetic comparative analyses. Contrasting patterns of chromosome number and genome size evolutions were detected between the two sister lineages. Mosses accumulated high chromosome number disparity via repeated whole‐genome duplications and descending dysploidy but maintained a small genome size. By contrast, the chromosome number of liverworts was highly conserved, and heterogeneous trends in genome size evolution were identified among major lineages. These contrasting patterns may be partly explained by the difference in genomic dynamics: Active dynamics enables genome downsizing and reorganization in mosses, whereas genome stability leads to the accumulation of large genomes in liverworts. The results of this study confirmed the distinct trends of genomic evolution in bryophytes.

Dual DNA replication modes: varying fork speeds and initiation rates within the spatial replication program in Xenopus.

Large vertebrate genomes duplicate by activating tens of thousands of DNA replication origins, irregularly spaced along the genome. The spatial and temporal regulation of the replication process is not yet fully understood. To investigate the DNA replication dynamics, we developed a methodology called RepliCorr, which uses the spatial correlation between replication patterns observed on stretched single-molecule DNA obtained by either DNA combing or high-throughput optical mapping. The analysis revealed two independent spatiotemporal processes that regulate the replication dynamics in the Xenopus model system. These mechanisms are referred to as a fast and a slow replication mode, differing by their opposite replication fork speed and rate of origin firing. We found that Polo-like kinase 1 (Plk1) depletion abolished the spatial separation of these two replication modes. In contrast, neither replication checkpoint inhibition nor Rap1-interacting factor (Rif1) depletion affected the distribution of these replication patterns. These results suggest that Plk1 plays an essential role in the local coordination of the spatial replication program and the initiation-elongation coupling along the chromosomes in Xenopus, ensuring the timely completion of the S phase.

Taming large-scale genomic analyses via sparsified genomics

Searching for similar genomic sequences is an essential and fundamental step in biomedical research. State-of-the-art computational methods performing such comparisons fail to cope with the exponential growth of genomic sequencing data. We introduce the concept of sparsified genomics where we systematically exclude a large number of bases from genomic sequences and enable faster and memory-efficient processing of the sparsified, shorter genomic sequences, while providing comparable accuracy to processing non-sparsified sequences. Sparsified genomics provides benefits to many genomic analyses and has broad applicability. Sparsifying genomic sequences accelerates the state-of-the-art read mapper (minimap2) by 2.57-5.38x, 1.13-2.78x, and 3.52-6.28x using real Illumina, HiFi, and ONT reads, respectively, while providing comparable memory footprint, 2x smaller index size, and more correctly detected variations compared to minimap2. Sparsifying genomic sequences makes containment search through very large genomes and large databases 72.7-75.88x (1.62-1.9x when indexing is preprocessed) faster and 723.3x more storage-efficient than searching through non-sparsified genomic sequences (with CMash and KMC3). Sparsifying genomic sequences enables robust microbiome discovery by providing 54.15-61.88x (1.58-1.71x when indexing is preprocessed) faster and 720x more storage-efficient taxonomic profiling of metagenomic samples over the state-of-the-art tool (Metalign).

Ecogenomic insights into the resilience of keystone Blastococcus Species in extreme environments: a comprehensive analysis

BackgroundThe stone-dwelling genus Blastococcus plays a key role in ecosystems facing extreme conditions such as drought, salinity, alkalinity, and heavy metal contamination. Despite its ecological significance, little is known about the genomic factors underpinning its adaptability and resilience in such harsh environments. This study investigates the genomic basis of Blastococcus's adaptability within its specific microniches, offering insights into its potential for biotechnological applications.ResultsComprehensive pangenome analysis revealed that Blastococcus possesses a highly dynamic genetic composition, characterized by a small core genome and a large accessory genome, indicating significant genomic plasticity. Ecogenomic assessments highlighted the genus's capabilities in substrate degradation, nutrient transport, and stress tolerance, particularly on stone surfaces and archaeological sites. The strains also exhibited plant growth-promoting traits, enhanced heavy metal resistance, and the ability to degrade environmental pollutants, positioning Blastococcus as a candidate for sustainable agriculture and bioremediation. Interestingly, no correlation was found between the ecological or plant growth-promoting traits (PGPR) of the strains and their isolation source, suggesting that these traits are not linked to their specific environments.ConclusionsThis research highlights the ecological and biotechnological potential of Blastococcus species in ecosystem health, soil fertility improvement, and stress mitigation strategies. It calls for further studies on the adaptation mechanisms of the genus, emphasizing the need to validate these findings through wet lab experiments. This study enhances our understanding of microbial ecology in extreme environments and supports the use of Blastococcus in environmental management, particularly in soil remediation and sustainable agricultural practices.

Genomic and Methylomic Signatures Associated With the Maintenance of Genome Stability and Adaptive Evolution in Two Closely Allied Wolf Spiders.

Pardosa spiders, belonging to the wolf spider family Lycosidae, play a vital role in maintaining the health of forest and agricultural ecosystems due to their function in pest control. This study presents chromosome-level genome assemblies for two allied Pardosa species, P. laura and P. agraria. Both species' genomes show a notable expansion of helitron transposable elements, which contributes to their large genome sizes. Methylome analysis indicates that P. laura has higher overall DNA methylation levels compared to P. agraria. DNA methylation may not only aids in transposable element-driven genome expansion but also positively affects the three-dimensional organisation of P. laura after transposon amplification, thereby potentially enhancing genome stability. Genes associated with hyper-differentially methylated regions in P. laura (compared to P. agraria) are enriched in functions related to mRNA processing and energy production. Furthermore, combined transcriptome and methylome profiling has uncovered a complex regulatory interplay between DNA methylation and gene expression, emphasising the important role of gene body methylation in the regulation of gene expression. Comparative genomic analysis shows a significant expansion of cuticle protein and detoxification-related gene families in P. laura, which may improve its adaptability to various habitats. This study provides essential genomic and methylomic insights, offering a deeper understanding of the relationship between transposable elements and genome stability, and illuminating the adaptive evolution and species differentiation among allied spiders.

The role of chromatin state in intron retention: A case study in leveraging large scale deep learning models.

Complex deep learning models trained on very large datasets have become key enabling tools for current research in natural language processing and computer vision. By providing pre-trained models that can be fine-tuned for specific applications, they enable researchers to create accurate models with minimal effort and computational resources. Large scale genomics deep learning models come in two flavors: the first are large language models of DNA sequences trained in a self-supervised fashion, similar to the corresponding natural language models; the second are supervised learning models that leverage large scale genomics datasets from ENCODE and other sources. We argue that these models are the equivalent of foundation models in natural language processing in their utility, as they encode within them chromatin state in its different aspects, providing useful representations that allow quick deployment of accurate models of gene regulation. We demonstrate this premise by leveraging the recently created Sei model to develop simple, interpretable models of intron retention, and demonstrate their advantage over models based on the DNA language model DNABERT-2. Our work also demonstrates the impact of chromatin state on the regulation of intron retention. Using representations learned by Sei, our model is able to discover the involvement of transcription factors and chromatin marks in regulating intron retention, providing better accuracy than a recently published custom model developed for this purpose.

Complete Genome Characterization of Canine Adenovirus From Türkiye With Next-Generation Sequencing.

Determining the complete genome sequence data of adenoviruses has recently become greatly important due to their use by scientists as vectors in cancer studies and other fields, including vaccine development. However, the GenBank database currently has few complete genome sequences of adenoviruses, which are known for their large genomes. To address this gap, we analysed next-generation sequencing data obtained from our previous study to provide the complete genome sequence of the canine adenovirus-2 strain. For the obtained canine adenovirus-2 strain (OQ596341), comparative genomics, recombination and phylogenetic analysis were conducted. This sequence was compared and phylogenetically analysed with the 20 complete genome sequences of canine adenovirus previously reported in GenBank worldwide, as well as partial E3 ORFA sequences obtained from Türkiye. The nucleotide similarity rates of the sequence obtained from this study with other CAdV-2 whole genomes are over 99.04%. The gene alignment results reveal that the OQ596341 was found to be closely related to the AC000020 reference genome and LC557011. There are two recombination events related to the genome in this study. Comparisons with other complete genome sequences revealed several previously unseen mutations. These mutations include H34Y in the E1A gene; P55A in the E1B 55K gene; D13N and D202N in the IVa2 gene; K679R, V934I and K989N in the Pol gene; E205K in the pTP gene; T455A in the pIIIa gene; A310V in the V gene; G151R in the protease gene; E268K in the 100K gene; G66S and G141S in the 33K gene; T14A, E250K, D287N and I293T in the E3 ORFA gene; and L193F in the E434K gene. Moreover, a comparison with partial sequences obtained from Türkiye revealed the E250K mutation in the E3 ORFA gene, which we report for the first time in Türkiye. The complete CAdV-2 genome sequence obtained in the present study is the first sequence from Europe. Comparative analysis with other genomes revealed some unique mutations. This study is the first to report the E250K amino acid change in the E3 ORFA gene in Türkiye. We anticipate that this data can be used in future CAdV-2 vaccine development studies. Further studies are recommended to evaluate the impact of these mutations on viral tropism and other host interactions.

Re-evaluating evidence for giant genomes in amoebae.

Here we reassess available evidence for the long-held misconception of amoebae possessing exceptionally large genomes. Traditionally, estimates relied on inaccurate methods like DNA weight measurements, leading to inflated sizes. These methods failed to account for contaminating DNA from prey, endosymbionts, and intrinsic genomic features like ribosomal operon amplification. Modern sequencing techniques unveil a different picture. Fully sequenced amoebozoa genomes range from 14.4 to 52.37 mega basepairs, well within the typical single-celled eukaryote expectation. While the whole genome of the historically relevant Amoeba proteus has not yet been fully sequenced, we provide here a statistical analysis using protein-coding genes from transcriptomic data, suggesting that the genome size is consistent with this range, far smaller than previously claimed. The misconception likely originated in the early 21st century and perpetuated through popular science materials. We conclude that there is no longer reason to reaffirm that amoeba genomes are giant.

Chromosome-scale genome assembly reveals how repeat elements shape non-coding RNA landscapes active during newt limb regeneration.

Newts have large genomes harboring many repeat elements. How these elements shape the genome and relate to newts' unique regeneration ability remains unknown. We present here the chromosome-scale assembly of the 20.3 Gb genome of the Iberian ribbed newt, Pleurodeles waltl, with a hitherto unprecedented contiguity and completeness among giant genomes. Utilizing this assembly, we demonstrate conserved synteny as well as genetic rearrangements, such as in the major histocompatibility complex locus. We provide evidence suggesting that intronic repeat elements drive newt-specific circular RNA (circRNA) biogenesis and show their regeneration-specific expression. We also present a comprehensive in-depth annotation and chromosomal mapping of microRNAs, highlighting genomic expansion profiles as well as a distinct regulatory pattern in the regenerating limb. These data reveal links between repeat elements, non-coding RNAs, and adult regeneration and provide key resources for addressing developmental, regenerative, and evolutionary principles.

Rnalib: a Python library for custom transcriptomics analyses.

The efficient and reproducible analysis of high-throughput sequencing datasets necessitates the development of methodical and robust computational pipelines that integrate established and bespoke bioinformatics analysis tools, often written in high-level programming languages such as Python. Despite the increasing availability of programming libraries for genomics, there is a noticeable lack of tools specifically focused on transcriptomics. Key tasks in this area include the association of gene features (e.g. transcript isoforms, introns or untranslated regions) with relevant subsections of (large) genomics datasets across diverse data formats, as well as efficient querying of these data based on genomic locations and annotation attributes. To address the needs of transcriptomics data analyses, we developed rnalib, a Python library designed for creating custom bioinformatics analysis methods. Built on existing Python libraries like pysam and pyBigWig, rnalib offers random access support, enabling efficient access to relevant subregions of large, genome-wide datasets. Rnalib extends the filtering and access capabilities of these libraries and includes additional checks to prevent common errors when integrating genomics datasets. The library is centred on an object-oriented Transcriptome class that provides methods for stepwise annotation of gene features with both, local and remote data sources. The rnalib Application Programming Interface cleanly separates immutable genomic locations from associated, mutable data, and offers a wide range of methods for iterating, querying, and exporting collated datasets. Rnalib establishes a fast, readable, reproducible, and robust framework for developing novel transcriptomics data analysis tools and methods. Source code, documentation, and tutorials are available at https://github.com/popitsch/rnalib.

Inter-chromosomal transcription hubs shape the 3D genome architecture of African trypanosomes

AbstractThe eukaryotic nucleus exhibits a highly organized 3D genome architecture, with RNA transcription and processing confined to specific nuclear structures. While intra-chromosomal interactions, such as promoter-enhancer dynamics, are well-studied, the role of inter-chromosomal interactions remains poorly understood. Investigating these interactions in mammalian cells is challenging due to large genome sizes and the need for deep sequencing. Additionally, transcription-dependent 3D topologies in mixed cell populations further complicate analyses. To address these challenges, we used high-resolution DNA-DNA contact mapping (Micro-C) in Trypanosoma brucei, a parasite with continuous RNA polymerase II (RNAPII) transcription and polycistronic transcription units (PTUs). With approximately 300 transcription start sites (TSSs), this genome organization simplifies data interpretation. To minimize scaffolding artifacts, we also generated a highly contiguous phased genome assembly using ultra-long sequencing reads. Our Micro-C analysis revealed an intricate 3D genome organization. While the T. brucei genome displays features resembling chromosome territories, its chromosomes are arranged around polymerase-specific transcription hubs. RNAPI-transcribed genes cluster, as expected from their localization to the nucleolus. However, we also found that RNAPII TSSs form distinct inter-chromosomal transcription hubs with other RNAPII TSSs. These findings highlight the evolutionary significance of inter-chromosomal transcription hubs and provide new insights into genome organization in T. brucei.

Optimising Exome Captures in Species With Large Genomes Using Species-Specific Repetitive DNA Blocker.

Large and highly repetitive genomes are common. However, research interests usually lie within the non-repetitive parts of the genome, as they are more likely functional, and can be used to answer questions related to adaptation, selection and evolutionary history. Exome capture is a cost-effective method for providing sequencing data from protein-coding parts of the genes. C0t-1 DNA blockers consist of repetitive DNA and are used in exome captures to prevent the hybridisation of repetitive DNA sequences to capture baits or bait-bound genomic DNA. Universal blockers target repetitive regions shared by many species, while species-specific c0t-1 DNA is prepared from the DNA of the studied species, thus perfectly matching the repetitive DNA contents of the species. So far, the use of species-specific c0t-1 DNA has been limited to a few model species. Here, we evaluated the performance of blocker treatments in exome captures of Pinus sylvestris, a widely distributed conifer species with a large (> 20 Gbp) and highly repetitive genome. We compared treatment with a commercial universal blocker to treatments with species-specific c0t-1 (30,000 and 60,000 ng). Species-specific c0t-1 captured more unique exons than the initial set of targets leading to increased SNP discovery and reduced sequencing of tandem repeats compared to the universal blocker. Based on our results, we recommend optimising exome captures using at least 60,000 ng of species-specific c0t-1 DNA. It is relatively easy and fast to prepare and can also be used with existing bait set designs.

Novel lineage of anelloviruses with large genomes identified in dolphins.

Anelloviruses are ubiquitous in mammals, but their infection has not yet been linked to any disease, suggesting a commensal virus-host relationship. Here, we describe the first anelloviruses associated with diverse species of dolphins. The dolphinid anelloviruses represent a new genus (tentatively named "Qoptorquevirus") and encode open reading frame 1 (ORF1) (capsid) proteins that are considerably larger than those encoded by previously described anelloviruses from other hosts. Comprehensive analysis of the ORF1 sequences and predicted protein structures revealed the underlying structural basis for such an extravagant ORF1 size and suggested that ORF1 size increased convergently in the anelloviruses associated with primate and Delphinidae hosts, respectively. Collectively, our results provide insights into the diversity and evolution of Anelloviridae. Further exploration of the anellovirus diversity, especially in the host species that have not yet been sampled, is expected to further clarify their evolutionary trajectory and explain the unusual virus-host commensal relationship.