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bioRxiv2022

MitoHiFi: a python pipeline for mitochondrial genome assembly from PacBio High Fidelity reads

BackgroundPacBio high fidelity (HiFi) sequencing reads are both long (15-20 kb) and highly accurate (>Q20). Because of these properties, they have revolutionised genome assembly leading to more accurate and contiguous genomes. In eukaryotes the mitochondrial genome is sequenced alongside the nuclear genome often at very high coverage. A dedicated tool for mitochondrial genome assembly using HiFi reads is still missing. ResultsMitoHiFi was developed within the Darwin Tree of Life Project to assemble mitochondrial genomes from the HiFi reads generated for target species. The input for MitoHiFi is either the raw reads or the assembled contigs, and the tool outputs a mitochondrial genome sequence fasta file along with annotation of protein and RNA genes. Variants arising from heteroplasmy are assembled independently, and nuclear insertions of mitochondrial sequences are identified and not used in organellar genome assembly. MitoHiFi has been used to assemble 374 mitochondrial genomes (369 from 12 phyla and 39 orders of Metazoa and from 6 species of Fungi) for the Darwin Tree of Life Project, the Vertebrate Genomes Project and the Aquatic Symbiosis Genome Project. Inspection of 60 mitochondrial genomes assembled with MitoHiFi for species that already have reference sequences in public databases showed the widespread presence of previously unreported repeats. ConclusionsMitoHiFi is able to assemble mitochondrial genomes from a wide phylogenetic range of taxa from Pacbio HiFi data. MitoHiFi is written in python and is freely available on github (https://github.com/marcelauliano/MitoHiFi). MitoHiFi is available with its dependencies as a singularity image on github (ghcr.io/marcelauliano/mitohifi:master).

bioinformatics↗

bioRxiv2023

Genome size and repeat content contribute to a complex architecture of flowering time in Amaranthus tuberculatus

Genome size variation, largely driven by repeat content, is poorly understood within and among populations, limiting our understanding of its significance for adaptation. Here we characterize intraspecific variation in genome size and repeat content across 186 individuals of Amaranthus tuberculatus, a ubiquitous native weed that shows flowering time adaptation to climate across its range and in response to agriculture. K-mer based genome size estimates vary by up to 20% across individuals, with transposable elements, unknown repeats, and rDNAs being the primary contributors to this variability. The additive effect of this variation has important phenotypic consequences--individuals with more repeats, and thus larger genomes, show slower flowering times and growth rates. Compared to newly-characterized gene copy number and polygenic nucleotide changes underlying variation in flowering time, we show that genome size remains a modest but significant contributor to the genetic basis of flowering time. Differences in flowering time across sexes and habitats are not mirrored by genome size variation, but rather polygenic variation and a gene copy number variant within the ATP synthesis pathway. Repeat content nonetheless shows non-neutral distributions across the genome, and across latitudinal and environmental gradients, reflecting numerous governing processes that in turn influence quantitative genetic variation for phenotypes key to plant adaptation. Author SummaryThe remarkable and seemingly inconsequential variation in genome size across species has long been an enigma in evolutionary biology. Calling this viewpoint into question, correlations between genome size variation and traits linked to fitness are increasingly uncovered. While this suggests that DNA content itself may be a source of adaptive genetic variation, repeat elements that propagate at the cost of the host are known to largely mediate this variation and may thus limit adaptive potential. Here we look to disentangle these multi-level dynamics, characterizing repeat dynamics across the genome and among individuals across diverse collections of a widespread agricultural weed, linking repeat content to genome size variation, and characterizing the relative importance of its phenotypic consequences. In Amaranthus tuberculatus, we find non-neutral repeat distributions across individuals across the range, and while this repeat variation underlies both variation in genome size and flowering time, we show that it makes a relatively minor contribution to variation in a fitness-related trait across the landscape relative to monogenic and polygenic features. Together, this work broadens our perspective on the complex selective dynamics that govern intraspecific variation in genome size and traits key to fitness in plants.

evolutionary biology↗

bioRxiv2023

TESOGENASE, An Engineered Nuclease Editor for Enhanced Targeted Genome Integration

Summary/AbstractNon-viral DNA donor template has been widely used for targeted genomic integration by homologous recombination (HR). This process has become more efficient with RNA guided endonuclease editor system such as CRISPR/Cas9. Circular single stranded DNA (cssDNA) has been harnessed previously as a genome engineering catalyst (GATALYST) for efficient and safe targeted gene knock-in. However, the engineering efficiency is bottlenecked by the nucleoplasm trafficking and genomic tethering of cssDNA donor, especially for extra-large transgene integration. Here we developed enGager, enhanced GATALYST associated genome editor system by fusion of nucleus localization signal (NLS) peptide tagged Cas9 with various single stranded DNA binding protein modules through a GFP reporter Knock-in screening. The enGager system assembles an integrative genome integration machinery by forming tripartite complex for engineered nuclease editors, sgRNA and ssDNA donors, thereby facilitate the nucleus trafficking of DNA donors and increase their active local concentration at the targeted genomic site. When applied for genome integration with cssDNA donor templates to diverse genomic loci in various cell types, these enGagers outperform unfused editors. The enhancement of integration efficiency ranges from 1.5- to more than 6-fold, with the effect being more prominent for > 4Kb transgene knock-in in primary cells. We further demonstrated that enGager mediated enhancement for genome integration is ssDNA, but less dsDNA dependent. Using one of the mini-enGagers, we demonstrated large chimeric antigen receptor (CAR) transgene integration in primary T cells with exceptional efficiency and anti-tumor function. These tripartite editors with ssDNA optimized genome engineering system (TESOGENASETM) add a set of novel endonuclease editors into the gene-editing toolbox for potential cell and gene therapeutic development based on ssDNA mediated non-viral genome engineering. HighlightO_LIA reporter Knock-in screening establishes enGager system to identify TESOGENASE editor to improving ssDNA mediated genome integration C_LIO_LIMini-TESOGENASEs developed by fusing Cas9 nuclease with novel ssDNA binding motifs C_LIO_LImRNA mini-TESOGENASEs enhance targeted genome integration via various non-viral delivery approaches C_LIO_LIEfficient functional CAR-T cell engineering by mini-TESOGENASE C_LI

bioengineering↗

bioRxiv2024

Genome streamlining to improve performance of a fast-growing cyanobacterium Synechococcus elongatus UTEX 2973

Cyanobacteria are photosynthetic organisms that have garnered significant recognition as potential hosts for sustainable bioproduction. However, their complex regulatory networks pose significant challenges to major metabolic engineering efforts, thereby limiting their feasibility as production hosts. Genome streamlining has been demonstrated to be a successful approach for improving productivity and fitness in heterotrophs but is yet to be explored to its full potential in phototrophs. Here we present the systematic reduction of the genome of the cyanobacterium exhibiting the fastest exponential growth, Synechococcus elongatus UTEX 2973. This work, the first of its kind in a photoautotroph, involved an iterative process using state-of-the-art genome-editing technology guided by experimental analysis and computational tools. CRISPR/Cas3 enabled large, progressive deletions of predicted dispensable regions and aided in the identification of essential genes. The large deletions were combined to obtain a strain with 55 kb genome reduction. The strains with streamlined genome showed improvement in growth (up to 23%) and productivity (by 22.7%) as compared to the WT. This streamlining strategy not only has the potential to develop cyanobacterial strains with improved growth and productivity traits but can also facilitate a better understanding of their genome to phenome relationships. ImportanceGenome streamlining is an evolutionary strategy used by natural living systems to dispense unnecessary genes from their genome as a mechanism to adapt and evolve. While this strategy has been successfully borrowed to develop synthetic heterotrophic microbial systems with desired phenotype, it has not been extensively explored in photoautotrophs. Genome streamlining strategy incorporates both computational predictions to identify the dispensable regions and experimental validation using genome editing tool and in this study we have employed a modified strategy with the goal to minimize the genome size to an extent that allows optimal cellular fitness under specified conditions. Our strategy has explored a novel genome-editing tool in photoautotrophs which, unlike other existing tools, enables large, spontaneous optimal deletions from the genome. Our findings demonstrate the effectiveness of this modified strategy in obtaining strains with streamlined genome, exhibiting improved fitness and productivity.

synthetic biology↗

bioRxiv2024

Chromosome-level genome assembly of the disco clam, Ctenoides ales, a first for the bivalve order Limida

The bivalve subclass Pteriomorphia, which includes the economically important scallops, oysters, mussels, and ark clams, exhibits extreme ecological, morphological, and behavioral diversity. Among this diversity are five morphologically distinct eye types, making Pteriomorphia an excellent setting to explore the molecular basis for the evolution of novel traits. Of pteriomorphian bivalves, Limida is the only order lacking genomic resources, greatly limiting the potential phylogenomic analyses related to eyes and phototransduction. Here, we present the first limid genome assembly, the disco clam, Ctenoides ales, which is characterized by invaginated eyes, exceptionally long tentacles, and a flashing light display. This genome assembly was constructed with PacBio long reads and Dovetail Omni-CTM proximity-ligation sequencing. The final assembly is [~]2.3Gb and over 99% of the total length is contained in 18 pseudomolecule scaffolds. We annotated 41,064 protein coding genes and report a BUSCO completeness of 91.9% for metazoa_obd10. Additionally, we report a completely annotated mitochondrial genome, also a first for Limida. The [~]20Kb mitogenome has 12 protein coding genes, 22 tRNAs, 2 rRNA genes, and a 1,589 bp duplicated sequence containing the origin of replication. The C. ales nuclear genome size is substantially larger than other pteriomorphian genomes, mainly accounted for by transposable element sequences. We inventoried the genome for opsins, the signaling proteins that initiate phototransduction, and found that, unlike its closest eyed-relatives, the scallops, C. ales lacks duplication of the rhabdomeric Gq-protein coupled opsin that is typically used for invertebrate vision. In fact, C. ales has uncharacteristically few opsins relative to the other pteriomorphian families, all of which have unique expansions of xenopsins, a recently discovered opsin subfamily. This chromosome-level assembly, along with the mitogenome, will be valuable resources for comparative genomics and phylogenetics in bivalves and particularly for the understudied but charismatic limids. SignificanceThis high-quality chromosome-level genome assembly for Ctenoides ales, the disco clam, is the first genome sequenced from the bivalve order Limida, the only group of Pteriomorphia - a highly diverse and ecologically important clade - lacking genomic resources. The sequence and annotation of the C. ales genome will be a useful resource for molluscan phylogenetics and comparative genomics.

evolutionary biology↗

bioRxiv2024

Global phylogenomic analysis of Staphylococcus pseudintermedius reveals genomic and prophage diversity in multi-drug resistant lineages

Staphylococcus pseudintermedius is the foremost cause of opportunistic canine skin and mucosal infections worldwide. Multidrug resistant (MDR) and methicillin-resistant S. pseudintermedius (MRSP) lineages have disseminated globally in the last decade and present significant treatment challenges. However, little is known regarding the factors that contribute to the success of MDR lineages. In this study, we compared the genome sequence of 110 UK isolates of S. pseudintermedius to 2,166 genomes of S. pseudintermedius populations from different continents. A novel core genome multi-locus typing scheme was generated to allow large scale, rapid and detailed analysis of S. pseudintermedius phylogenies, and was used to show that the S. pseudintermedius population structure is broadly segregated into an MDR population and a non-MDR population. MRSP lineages are predicted to either encode certain resistance genes chromosomally, or on plasmids, and this is associated with their MLST sequence type. Comparison of lineages most frequently implicated in disease, ST-45 and ST-71, with the phylogenetically related ST-496 lineage that has a comparatively low disease rate, revealed that ST-45 and ST-71 genomes encode distinct combinations of phage-defence systems and concurrently encode a high number of intact prophages. In contrast, ST-496 genomes encode a wider array of phage defence systems and lack intact, complete prophages. Additionally, we show that distinct prophages are widespread in S. pseudintermedius and appear to account for the vast majority of genomic diversity. These findings indicate that MRSP lineages have significant structural genomic differences, and that prophage integration, and differential antiviral systems correlate with the emergence of successful genotypes. Impact StatementStaphylococcus pseudintermedius is a major cause of soft tissue infections in dogs but may occasionally infect other companion animals and humans, most often veterinary professionals in primary care. Methicillin-resistant Staphylococcus pseudintermedius (MRSP) have emerged worldwide and are often linked to resistance to multiple antimicrobials, resulting in a significant health burden. Here, we analysed a large collection of S. pseudintermedius genome sequences, which has allowed a detailed characterisation of the molecular epidemiology and diversity of the species using a novel typing scheme. Here we show that closely related MRSP lineages differ in whether specific antibiotic resistance genes are encoded on potentially mobilisable plasmids, or more stably on the chromosome, indicating differing evolutionary trajectories of MRSP lineages. Comparison of the S. pseudintermedius types most frequently implicated in clinical cases to those associated with commensal carriage, showed that known genes thought to contribute to disease are universal, and therefore, not associated with the high incidence rates of disease of particular lineages. We demonstrate that MRSP sequence types linked to high disease rates lack specific phage-defence systems and are associated with a high burden of prophages. Data statementAll supporting data, code and protocols have been provided either within the article, through supplementary data files, or via Figshare and Zenodo. Six supplementary figures and one supplementary file are available with the online version of this article. Data summaryAll genome sequencing data (sequencing reads and genome assemblies) are available at the Sequence Read Archive (SRA) and Genomes repositories at NCBI, in Bioproject number PRJNA1153484. The information for the individual S. pseudintermedius genomes has been included in Supplementary File 1. Genome assemblies used in this study are available from https://zenodo.org/records/13692319, DOI 10.5281/zenodo.13692318. The S. pseudintermedius cgMLST scheme developed for this study has been made available via Zenodo (https://zenodo.org/records/13633136, DOI: 10.5281/zenodo.13633135) and Figshare (https://doi.org/10.6084/m9.figshare.26911654.v1, DOI: 10.6084/m9.figshare.26911654).

microbiology↗

bioRxiv2024

Re-calibration of flow cytometry standards for plant genome size estimation

The evolution of long-read sequencing technologies has advanced the development of genome assemblies that are now frequently presented as gap-free or nearly gapless, reflecting substantial improvements in sequencing accuracy and completeness. However, discrepancies between the genome size estimates derived from genome assemblies and flow cytometry create ambiguity regarding the accuracy of these complementary approaches. Accurate genome size estimation via flow cytometry relies on use of an internal standard with a genome of known size. Historically, the genome size of these standards was often calibrated against incomplete genome assemblies or non-plant genomes, such as the human male genome, which was previously considered to be 7 pg but is now known to be around 6.15 pg after 20 years of advancements. Calibrating plant references against non-plant standards is not recommended due to differential staining properties. Therefore, we recalibrated the size of five plant genomes commonly used as reference standards in flow cytometry, by utilizing a recent gapless, telomere-to-telomere (T2T) genome assembly of Nipponbare rice. Our results indicate a significant overestimation of around 20% in previous flow cytometry-based estimates for Pisum sativum and Nicotiana benthamiana, around 10% for Arabidopsis thaliana, and less than 5% for Sorghum bicolor, and Gossypium hirsutum. The close alignment of the recalibrated GS estimates to the reference genome assemblies and recalculated estimates from different studies confirms their suitability as reference standards for more accurate measurement of plant genome size.

molecular biology↗

bioRxiv2024

The challenge of sequencing Chlamydia trachomatis and other bacterial STI genomes directly from clinical swabs: the optimum solution

Rates of bacterial sexually transmitted infections (STIs) are rising and accessing their genomes provides information on strain evolution, circulating strains, and encoded antimicrobial resistance (AMR). Notable pathogens include Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Treponema pallidum (TP), globally the most common bacterial STIs. Mycoplasma genitalium (MG) is also a bacterial STI which is of concern due to AMR development. These bacteria are also fastidious or hard to culture, and standard sampling methods lyse bacteria, completely preventing pathogen culture. Clinical samples contain large amounts of human and other microbiota DNA. These factors hinder the sequencing of bacterial STI genomes. We aimed to overcome these challenges in obtaining whole genome sequences, and evaluated four approaches using clinical samples from Argentina (39), Switzerland (14), and cultured samples from Finland (2) and Argentina (1). First, direct genome sequencing from swab samples was attempted through Illumina deep metagenomic sequencing, showing extremely low levels of target DNA, with under 0.01% of the sequenced reads being from the target pathogens. Second, host DNA depletion followed by Illumina sequencing was not found to produce enrichment in these very low load samples. Third, we tried a selective long-read approach with the new adaptive sequencing from Oxford Nanopore Technologies (ONT), which also did not improve enrichment sufficiently to provide genomic information. Finally, target enrichment using a novel pan-genome set of custom SureSelect probes targeting CT, NG, TP, and MG followed by Illumina sequencing was successful. We produced whole genomes from 64% of CT positive samples; from 36% of NG positive samples, and from 60% of TP positive samples. Additionally, we enriched MG DNA to gain partial genomes from 60% of samples. This is the first publication to date to utilize a pan-genome STI panel in target enrichment. Target enrichment, though costly, proved essential for obtaining genomic data from clinical samples. This data can be utilized to examine circulating strains, genotypic resistance, and guide public health strategies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/624631v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@578aeborg.highwire.dtl.DTLVardef@16185f9org.highwire.dtl.DTLVardef@1a2ae87org.highwire.dtl.DTLVardef@1703fa7_HPS_FORMAT_FIGEXP M_FIG C_FIG Impact statementGenome data on circulating sexually transmitted infections (STIs) is important to better understand transmission networks, antimicrobial resistance and to guide treatment decisions. For many bacterial STIs, this information is difficult to obtain, as the bacteria are fastidious, in some cases intracellular, and often recalcitrant to culture. We have developed and tested a target enrichment STI panel of baits to capture whole genomes of Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, and Mycoplasma genitalium with approximately 50% success in genome sequencing for the first three pathogens. We compare this against other sequencing and enrichment methods, which did not provide sufficient data for genome analysis. This panel approach shows potential for clinical samples carrying these pathogens and can potentially also be developed for further pathogen groups. Data summaryAll illumina sequence data, with human read data removed using Hostile (1) and KrakenTools (https://github.com/jenniferlu717/KrakenTools), is deposited with the European Nucleotide Archive (ENA) under project number PRJEB72167.

microbiology↗

bioRxiv2025

seqLens: optimizing language models for genomic predictions

Understanding evolutionary variation in genomic sequences through the lens of language modeling has the potential to revolutionize biological research. Yet to maximize the utility of language modeling in genomics, we must overcome computational challenges in tokenization and model architecture adapted to diverse genomic features across evolutionary timescales. In this study, we investigated key elements in genomic language modeling (gLM), including tokenization, pretraining datasets, fine-tuning approaches, pooling methods, and domain adaptation, and applied the language models to diverse genomic data. We gathered two evolutionarily distinct pretraining datasets: one consisting of 19,551 reference genomes, including over 18,000 prokaryotic genomes (115B nucleotides) and the remainder eukaryotic genomes, and another more balanced dataset with 1,354 genomes, including 1,166 prokaryotic and 188 eukaryotic reference genomes (180B nucleotides). We trained five byte-pair encoding tokenizers and pretrained 52 gLMs, systematically comparing different architectures, hyperparameters, and classification heads. We introduce seqLens, a family of models based on disentangled attention with relative positional encoding, which outperforms relatively similar-sized models in 13 of 19 benchmarking phenotypic predictions. We further explore continual pretraining, domain adaptation, and parameter-efficient fine-tuning methods to assess trade-offs between computational efficiency and accuracy. Our findings demonstrate that relevant pretraining data significantly boost performance, alternative pooling techniques can enhance classification, tokenizers with larger vocabulary sizes negatively impact generalization, and gLMs are capable of understanding evolutionary relationships. These insights provide a foundation for optimizing genomic language models for identifying diverse evolutionary genomic features and improving genome annotations.

bioinformatics↗

bioRxiv2025

The path to dependence: stepwise genome evolution from a facultative symbiont to an endosymbiont in the N2-fixing diatom-Richelia symbioses

A few genera of diatoms are widespread in the oceans and form stable partnerships with N2-fixing filamentous cyanobacteria Richelia spp. A unique feature of the diatom-Richelia symbioses is the symbiont cellular location spans a continuum of integration (epibiont, periplasmic, endobiont) that is reflected in the symbiont genome size and content. In this study we analyzed genomes derived from cultures and environmental metagenome-assembled genomes focusing on characters indicative of genome evolution. Our results show an enrichment of short length transposable elements (TEs) and pseudogenes in the periplasmic endosymbiont genomes, suggesting an active and transitionary period in genome evolution. In contrast, genomes of endobionts exhibited fewer TEs and pseudogenes, reflecting advanced stages of genome reduction and increased host dependency in the endobionts. Pangenome analyses identified that endobionts streamline their genomes and conserve a majority of their genes in the core, whereas periplasmic endobionts and epibionts maintain larger flexible genomes, suggesting increased genomic plasticity. Functional gene comparisons with other N2-fixing cyanobacteria revealed that Richelia endobionts have similar patterns of metabolic loss but are distinguished by absence of specific pathways (e.g. cytochrome bd ubiquinol oxidase, lipid-A) that increase dependency and direct interaction with their respective hosts. In conclusion, our findings underscore the dynamic nature of genome reduction in N2-fixing cyanobacterial symbionts and demonstrate the diatom-Richelia symbioses as a valuable and rare model to study genome evolution in the transitional stages from a free-living facultative symbiont to a host-dependent endobiont.

microbiology↗

bioRxiv2025

Life at the extremes: Maximally divergent microbes with similar genomic signatures linked to extreme environments

Extreme environments impose strong mutation and selection pressures that drive distinctive, yet understudied, genomic adaptations in extremophiles. In this study, we identify 15 bacterium-archaeon pairs that exhibit highly similar k-mer-based genomic signatures despite maximal taxonomic divergence, suggesting that shared environmental conditions can produce convergent, genome-wide patterns that transcend evolutionary distance. To uncover these patterns, we developed a computational pipeline to select a composite genome proxy assembled from non-contiguous subsequences of the genome. Using supervised machine learning on a curated dataset of 693 extremophile microbial genomes, we found that 6-mers and 100 kbp genome proxy lengths provide the best balance between classification accuracy and computational efficiency. Our results provide conclusive evidence of the pervasive nature of k-mer-based patterns across the genome, and uncover the presence of taxonomic and environmental components that persist across all regions of the genome. The 15 bacterium-archaeon pairs identified by our method as having similar genomic signatures were validated through multiple independent analyses, including 3-mer frequency profile comparisons, phenotypic trait similarity, and geographic co-occurrence data. These complementary validations confirmed that extreme environmental pressures can override traditionally recognized taxonomic components at the whole-genome level. Together, these findings reveal that adaptation to extreme conditions can carry robust, taxonomic domain-spanning imprints on microbial genomes, offering new insight into the relationship between environmental mutagenesis and selection and genome-wide evolutionary convergence.

bioinformatics↗

bioRxiv2018

Ecological selection for small microbial genomes along a temperate-to-thermal soil gradient

Small bacterial and archaeal genomes provide insights into the minimal requirements for life1 and seem to be widespread on the microbial phylogenetic tree2. We know that evolutionary processes, mainly selection and drift, can result in microbial genome reduction 3,4. However, we do not know the precise environmental pressures that constrain genome size in free-living microorganisms. A study including isolates 5 has shown that bacteria with high optimum growth temperatures, including thermophiles, often have small genomes 6. It is unclear how well this relationship may extend generally to microorganisms in nature 7,8, and in particular to those microbes inhabiting complex and highly variable environments like soil 3,6,9. To understand the genomic traits of thermally-adapted microorganisms, here we investigated bacterial and archaeal metagenomes from a 45{degrees}C gradient of temperate-to-thermal soils overlying the ongoing Centralia, Pennsylvania (USA) coal seam fire. There was a strong relationship between average genome size and temperature: hot soils had small genomes relative to ambient soils (Pearsons r = -0.910, p < 0.001). There was also an inverse relationship between soil temperature and cell size (Pearsons r = -0.65, p = 0.021), providing evidence that cell and genome size in the wild are together constrained by temperature. Notably, hot soils had different community structures than ambient soils, implicating ecological selection for thermo-tolerant cells that had small genomes, rather than contemporary genome streamlining within the local populations. Hot soils notably lacked genes for described two-component regulatory systems and antimicrobial production and resistance. Our work provides field evidence for the inverse relationship between microbial genome size and temperature requirements in a diverse, free-living community over a wide range of temperatures that support microbial life. Our findings demonstrate that ecological selection for thermophiles and thermo-tolerant microorganisms can result in smaller average genome sizes in situ, possibly because they have small genomes reminiscent of a more ancestral state.

microbiology↗

bioRxiv2018

Thermosipho spp. immune system differences affect variation in genome size and geographical distributions

Thermosipho species inhabit thermal environments such as marine hydrothermal vents, petroleum reservoirs and terrestrial hot springs. A 16S rRNA phylogeny of available Thermosipho spp. sequences suggested habitat specialists adapted to living in hydrothermal vents only, and habitat generalists inhabiting oil reservoirs, hydrothermal vents and hotsprings. Comparative genomics of 15 Thermosipho genomes separated them into three distinct species with different habitat distributions: the widely distributed T. africanus and the more specialized, T. melanesiensis and T. affectus. Moreover, the species can be differentiated on the basis of genome size, genome content and immune system composition. For instance, the T. africanus genomes are largest and contained the most carbohydrate metabolism genes, which could explain why these isolates were obtained from ecologically more divergent habitats. Nonetheless, all the Thermosipho genomes, like other Thermotogae genomes, show evidence of genome streamlining. Genome size differences between the species could further be correlated to differences in defense capacities against foreign DNA, which influence recombination via HGT. The smallest genomes are found in T. affectus that contain both CRISPR-cas Type I and III systems, but no RM system genes. We suggest that this has caused these genomes to be almost devoid of mobile elements, contrasting the two other species genomes that contain a higher abundance of mobile elements combined with different immune system configurations. Taken together, the comparative genomic analyses of Thermosipho spp. revealed genetic variation allowing habitat differentiation within the genus as well as differentiation with respect to invading mobile DNA.

microbiology↗

bioRxiv2018

Genomic differentiation is initiated without physical linkage among targets of divergent selection in Fall armyworms

The process of speciation involves whole genome differentiation by overcoming gene flow between diverging populations. We have ample knowledge which evolutionary forces may cause genomic differentiation, and several speciation models have been proposed to explain the transition from genetic to genomic differentiation. However, it is still unclear what are critical conditions enabling genomic differentiation in nature. The Fall armyworm, Spodoptera frugiperda, is observed as two sympatric strains that have different host-plant ranges, suggesting the possibility of ecological divergent selection. In our previous study, we observed that these two strains show genetic differentiation across the whole genome with an unprecedentedly low extent, suggesting the possibility that whole genome sequences started to be differentiated between the strains. In this study, we analyzed whole genome sequences from these two strains from Mississippi to identify critical evolutionary factors for genomic differentiation. The genomic Fst is low (0.017) while 91.3% of 10kb windows have Fst greater than 0, suggesting genome-wide differentiation with a low extent. We identified nearly 400 outliers of genetic differentiation between strains, and found that physical linkage among these outliers is not a primary cause of genomic differentiation. Fst is not significantly correlated with gene density, a proxy for the strength of selection, suggesting that a genomic reduction in migration rate dominates the extent of local genetic differentiation. Our analyses reveal that divergent selection alone is sufficient to generate genomic differentiation, and any following diversifying factors may increase the level of genetic differentiation between diverging strains in the process of speciation.

evolutionary biology↗

bioRxiv2019

Horizontal transmission and recombination maintain forever young bacterial symbiont genomes

Bacterial symbionts bring a wealth of functions to the associations they participate in, but by doing so, they endanger the genes and genomes underlying these abilities. When bacterial symbionts become obligately associated with their hosts, their genomes are thought to decay towards an organelle-like fate due to decreased homologous recombination and inefficient selection. However, numerous associations exist that counter these expectations, especially in marine environments, possibly due to ongoing horizontal gene flow. Despite extensive theoretical treatment, no empirical study thus far has connected these underlying population genetic processes with long-term evolutionary outcomes. By sampling marine chemosynthetic bacterial-bivalve endosymbioses that range from primarily vertical to strictly horizontal transmission, we tested this canonical theory. We found that transmission mode strongly predicts homologous recombination rates, and that exceedingly low recombination rates are associated with moderate genome degradation in the marine symbionts with nearly strict vertical transmission. Nonetheless, even the most degraded marine endosymbiont genomes are occasionally horizontally transmitted and are much larger than their terrestrial insect symbiont counterparts. Therefore, horizontal transmission and recombination enable efficient natural selection to maintain intermediate symbiont genome sizes and substantial functional genetic variation. Author summarySymbiotic associations between bacteria and eukaryotes are ubiquitous in nature and have contributed to the evolution of radically novel phenotypes and niches for the involved partners. New metabolic or physiological capacities that arise in these associations are typically encoded by the bacterial symbiont genomes. However, the association itself endangers the retention of bacterial genomic coding capacity. Endosymbiont genome evolution theory predicts that when bacterial symbionts become restricted to host tissues, their populations cannot remove deleterious mutations efficiently. This ultimately results in their genomes degrading to small, function-poor states, reminiscent of organellar genomes. However, many ancient marine endosymbionts do not fit this prediction, but instead retain relatively large, gene-rich genomes, indicating that the evolutionary dynamics of this process need more thorough characterization. Here we show that on-going symbiont gene flow via horizontal transmission between bivalve hosts and recombination among divergent gammaproteobacterial symbiont lineages are sufficient to maintain large and dynamic bacterial symbiont genomes. These findings indicate that many obligately associated symbiont genomes may not be as isolated from one another as previously assumed and are not on a one way path to degradation.

evolutionary biology↗

bioRxiv2019

Microrheology for Hi-C Data Reveals the Spectrum of the Dynamic 3D Genome Organization

The 1-dimensional information of genomic DNA is hierarchically packed inside the eukaryotic cell nucleus and organized in 3-dimensional (3D) space. Genome-wide chromosome conformation capture (Hi-C) methods have uncovered the 3D genome organization and revealed multiscale chromatin domains of compartments and topologically associating domains (TADs). Moreover, single-nucleosome live-cell imaging experiments have revealed the dynamic organization of chromatin domains caused by stochastic thermal fluctuations. However, the mechanism underlying the dynamic regulation of such hierarchical and structural chromatin units within the micro-scale thermal medium remains unclear. Microrheology is a way to measure dynamic viscoelastic properties coupling between thermal microenvironment and mechanical response. Here, we propose, to our knowledge, a new microrheology for Hi-C data to analyze the compliance property as a barometer of rigidness and flexibility of genomic regions along with the time evolution. Our method allows conversion of a Hi-C matrix into the spectrum of the rheological property along the genomic coordinate of a single chromosome. To demonstrate the technique, we analyzed Hi-C data during the neural differentiation of mouse embryonic stem cells. We found that TAD boundaries behave as more rigid nodes than the intra-TAD region. The spectrum clearly shows the rheological property of the dynamic chromatin domain formations at an individual time scale. Furthermore, we characterized the appearance of synchronous and liquid-like inter-compartment interactions in differentiated cells. Together, our microrheology provides physical insights revealing the dynamic 3D genome organization from Hi-C data.\n\nSIGNIFICANCEGenomic DNA is hierarchically packed inside the eukaryotic cell nucleus, and the genome organization in 3D contributes to proper genome functions at the multiscale chromatin domains. Although thermal fluctuations inevitably drive movements of the genome molecules in the micro-scale cell environment, there is no method, as yet, to quantify such dynamic 3D genome organization of hierarchical and structural chromatin units. Here, we describe a method to calculate rheological properties as barometers of flexibility and liquid-like behavior of genomic regions. We show that biologically relevant boundaries between chromatin domains are more rigid than the inside at a particular time scale. Our method allows interpretation of static and population-averaged genome conformation data as dynamic and hierarchical 3D genome picture.

biophysics↗

bioRxiv2025

Wavelet Based Whole Genome Doubling Aware Single Cell Copy Number Calling

Advances in single cell whole genome sequencing enable profiling of the copy number state of thousands of cells with minimal sequencing bias across the genome. The Direct Library Preparation + technique is an whole genome amplification-free single cell whole genome sequencing method that achieves high throughput by fragmenting each cells genome and ligating sequencing adapters using a modified Tn5 transposase, and sequencing to less than 0.1x coverage. Despite recent advances in experimental approaches, data analysis of single cell whole genome sequencing lags behind and the existing methods are not optimized for the analysis of frozen samples with variable DNA preservation. Furthermore, existing tools predominantly rely on read depth ratio in predefined genomic bins to call copy number, making whole genome duplication unidentifiable. To address this, we introduce Songbird, a single cell whole genome sequencing copy number caller that is whole genome duplication sensitive, and outperforms existing tools both in breakpoints identification and true copy number detection. We demonstrate that Songbird is robust down to extremely low coverage, adaptable to a variety of genome versions (hg19, hg38, hs.1), and is extensible to other single cell whole genome sequencing methods that rely on Tn5 tagmentation to fragment the genome.

bioinformatics↗

bioRxiv2026

Reference genome assembly of the tetraploid tuber crop Tropaeolum tuberosum

Tropaeolum tuberosum is a tuber-forming crop native to the Andes, valued for its nutritional content, pest resistance and adaptation to high-altitude environments. Despite its contribution to food security and sustainable agriculture, it remains an underutilized species with scarce genomic resources, limiting genetic and functional studies. To address this gap, we generated a reference genome assembly for a European ex situ tetraploid accession of T. tuberosum using PacBio HiFi sequencing. The assembly spans 1.3 Gb in 1,805 contigs (contigs N50 = 32.2 Mb, longest contig = 60 Mb) and recovers 87% of the estimated genome size. We assessed assembly completeness and accuracy using Benchmarking Universal Single-Copy Orthologs, which detected 98.5% complete genes (21.7% single-copy, 76.8% duplicated), 0.7% fragmented, and 0.7% missing, demonstrating near-complete gene space recovery consistent with a high-quality tetraploid genome. To evaluate transferability, we resequenced a field-collected Colombian genotype of economic relevance using Oxford Nanopore technology. Read mapping showed 99.7% of primary alignments to the reference (weighted mean coverage = 16.4x, 96.1% at [≥]5x), confirming broad sequence conservation between accessions and validating the suitability of the ex situ reference as an anchor for in situ germplasm. Repetitive elements accounted for 71.3% of the genome. Using ANNEVO, we annotated 56,354 high-confidence protein-coding genes, achieving 98.3% complete BUSCOs, a PSAURON score of 97.2, and 90.5% taxonomic consistency with the rosid lineage (OMArk). This high-quality reference genome establishes a foundational resource for comparative genomics, population genomics and functional analyses on T. tuberosum, supporting future breeding and conservation of this important food resource. Significance statementT. tuberosum is an underutilized Andean tuber crop that combines highly nutritious tubers with remarkable disease resistance and UV tolerance, supporting Andean smallholder livelihoods under harsh high-altitude conditions. Beyond its role as a food crop, it is cultivated widely as an ornamental due to its abundant, brightly colored flowers closely resembling those of its relative nasturtium. Despite this dual relevance, genomic resources have been entirely lacking, limiting efforts to understand its domestication history, adaptation, and potential as a climate-resilient alternative to potato with higher nutraceutical properties. Our reference genome assembly provides the first genome-scale resource for T. tuberosum, creating a foundation for dissecting polyploid genome organization, key agronomic and nutritional traits, and interactions with biotic and abiotic stress. By enabling evolution, domestication, comparative genomics, breeding, conservation, and functional studies, this resource unlocks T. tuberosum as a model for resilient, diversified cropping systems globally.

plant biology↗