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bioRxiv2021

Evolution of genome fragility enables microbial division of labor

Division of labor can evolve when social groups benefit from the functional specialization of its members. Recently, a novel means of coordinating division of labor was found in the antibiotic-producing bacterium Streptomyces coelicolor, where functionally specialized cells are generated through large-scale genomic re-organization. Here, we investigate how the evolution of a genome architecture enables such mutation-driven division of labor, using a multi-scale mathematical model of bacterial evolution. We let bacteria compete on the basis of their antibiotic production and growth rate in a spatially structured environment. Bacterial behavior is determined by the structure and composition of their genome, which encodes antibiotics, growth-promoting genes and fragile genomic loci that can induce chromosomal deletions. We find that a genomic organization evolves that partitions growth-promoting genes and antibiotic-coding genes to distinct parts of the genome, separated by fragile genomic loci. Mutations caused by these fragile sites mostly delete growth-promoting genes, generating antibiotic-producing mutants from non-producing (and weakly-producing) progenitors, in agreement with experimental observations. Mutants protect their colony from competitors but are themselves unable to replicate. We further show that this division of labor enhances the local competition between colonies by promoting antibiotic diversity. These results show that genomic organization can co-evolve with genomic instabilities to enable reproductive division of labor. Motivation of current workDivision of labor can evolve if trade-offs are present between different traits. To organize a division of labor, the genome architecture must evolve to enable differentiated cellular phenotypes. Cell differentiation may be coordinated through gene regulation, as occurs during embryonic development. Alternatively, when mutation rates are high, mutations themselves can guide cell and functional differentiation; however, how this evolves and is organized at the genome level remains unclear. Here, using a model of antibiotic-producing bacteria based on multicellular Streptomyces, we show that if antibiotic production trades off with replication, genome architecture can evolve to support a mutation-driven division of labor. These results are consistent with recent experimental observations and may underlie division of labor in many bacterial groups.

evolutionary biology↗
bioRxiv2021

Genome-wide analysis of long terminal repeat retrotransposons from the cranberry Vaccinium macrocarpon

BACKGROUNDLong terminal repeat (LTR) retrotransposons are widespread in plant genomes and play a large role in the generation of genomic variation. Despite this, their identification and characterization remains challenging, especially for non-model genomes. Hence, LTR retrotransposons remain undercharacterized in Vaccinium genomes, although they may be beneficial for current berry breeding efforts. OBJECTIVEExemplarily focusing on the genome of American cranberry (Vaccinium macrocarpon Aiton), we aim to generate an overview of the LTR retrotransposon landscape, highlighting the abundance, transcriptional activity, sequence, and structure of the major retrotransposon lineages. METHODSGraph-based clustering of whole genome shotgun Illumina reads was performed to identify the most abundant LTR retrotransposons and to reconstruct representative in silico full-length elements. To generate insights into the LTR retrotransposon diversity in V. macrocarpon, we also queried the genome assembly for presence of reverse transcriptases (RTs), the key domain of LTR retrotransposons. Using transcriptomic data, transcriptional activity of retrotransposons corresponding to the consensuses was analyzed. RESULTSWe provide an in-depth characterization of the LTR retrotransposon landscape in the V. macrocarpon genome. Based on 475 RTs harvested from the genome assembly, we detect a high retrotransposon variety, with all major lineages present. To better understand their structural hallmarks, we reconstructed 26 Ty1-copia and 28 Ty3-gypsy in silico consensuses that capture the detected diversity. Accordingly, we frequently identify association with tandemly repeated motifs, extra open reading frames, and specialized, lineage-typical domains. Based on the overall high genomic abundance and transcriptional activity, we suggest that retrotransposons of the Ale and Athila lineages are most promising to monitor retrotransposon-derived polymorphisms across accessions. CONCLUSIONSWe conclude that LTR retrotransposons are major components of the V. macrocarpon genome. The representative consensuses provide an entry point for further Vaccinium genome analyses and may be applied to derive molecular markers for enhancing cranberry selection and breeding.

plant biology↗
bioRxiv2021

Genetic Characterization and Curation of Diploid A-Genome Wheat Species

The A-genome diploid wheats represent the earliest domesticated and cultivated wheat species in the Fertile Crescent and the donor of the wheat A sub-genome. The A-genome species encompass the cultivated einkorn (Triticum. monococcum L. subsp. monococcum), wild einkorn (T. monococcum L. subsp. aegilopoides (Link) Thell.) and T. urartu. We evaluated the collection of 930 accessions in the Wheat Genetics Resource Center (WGRC), using genotyping-by-sequencing (GBS) and identified 13,089 curated SNPs. Genomic analysis detected misclassified and duplicated accessions with most duplicates originated from the same or a nearby locations. About 56% (n = 520) of the WGRC A-genome species collections were duplicates supporting the need for genomic characterization for effective curation and maintenance of these collections. Population structure analysis confirmed the morphology-based classifications of the accessions and reflected the species geographic distributions. We also showed that the T. urartu as the closest A-genome diploid to wheat through phylogenetic analysis. Population analysis within the wild einkorn group showed three genetically distinct clusters, which corresponded with wild einkorn races , {beta}, and {gamma} described previously. The T. monococcum genome-wide FST scan identified candidate genomic regions harboring domestication selection signature (Btr1) on the short arm of chromosome 3Am at ~ 70 Mb. We established A-genome core set (79 accessions) based on allelic diversity, geographical distribution, and available phenotypic data. The individual species core set maintained at least 80% of allelic variants in the A-genome collection and constitute a valuable genetic resource to improve wheat and domesticated einkorn in breeding programs. One-sentence summaryGenotyping of gene bank collections of diploid A-genome relatives of wheat uncovered relatively higher genetic diversity and unique evolutionary relationships which gives insight to the effective use of these germplasm for wheat improvement.

genetics↗
bioRxiv2022

diemr: Genome polarisation for detecting barriers to geneflow in R

O_LISemi-permeable barriers to geneflow in principle allow distantly related organisms to capture and exchange pre-adapted genes potentially speeding adaptation. However, describing barriers to geneflow on a genomic scale is non-trivial. C_LIO_LIWe extend classic diagnostic allele counting measures of geneflow across a barrier to the case of genome-scale data. Diagnostic index expectation maximisation (diem) polarises the labelling of bistate markers with respect to the sides of a barrier. An initial state of ignorance is enforced by starting with randomly generated marker polarisations. This means there is no prior on population or taxon membership of the genomes concerned. Using a deterministic data labelling, small numbers of classic diagnostic markers can be replaced by large numbers of markers, each with a diagnostic index. Individuals hybrid indices (genome admixture proportions) are then calculated genome wide conditioned on marker diagnosticity; within diploid, haplodiploid and/or haploid genome compartments; or indeed over any subset of markers, allowing classical cline width/barrier strength comparisons along genomes. Along-genome barrier strength hetero-geneity allows for barrier regions to be identified. Further, blocks of genetic material that have introgressed across a barrier are easily identified with high power. C_LIO_LIdiem indicates panmixis among Myotis myotis bat genomes, with a barrier separating low data quality outliers. In a Mus musculus domesticus/Mus spretus system, diem adds multiple introgressions of olfactory (and vomeronasal) gene clusters in one direction to previous demon-strations of a pesticide resistance gene introgressing in the opposite direction across a strong species barrier. C_LIO_LIdiem is a genomes analysis solution which scales over reduced representation genomics of thousands of markers to treatment of all variant sites in large genomes. While the method lends itself to visualisation, its output of markers with barrier-informative annotation will fuel research in population genetics, phylogenetics and association studies. diem can equip such downstream applications with millions of informative markers. C_LI

bioinformatics↗
bioRxiv2022

Apurinic/apyrimidinic nuclease 1 drives genomic evolution contributing to chemoresistance and tumorigenesis in solid tumor

Genomic instability fuels genomic alterations that befit cancer cells with necessary adaptations to keep proliferating and overcome the impact of host anti-tumor immunity and cytotoxic therapy. Since DNA breaks are required for genomic rearrangements to take place, we hypothesized that dysregulated nuclease activity mediates genomic instability in cancer. Using an integrated genomics protocol, we identified a four gene deoxyribonuclease signature correlating with genomic instability in six human cancers which included adenocarcinomas of esophagus (EAC), lung, prostate, stomach, pancreas and triple negative breast cancer. Functional screens confirmed the role of these nucleases in genomic instability and growth of cancer cells. Apurinic/apyrimidinic nuclease 1 (APE1), identified as top nuclease in functional screen, was further investigated in five cell lines representing four solid tumors (EAC, lung, prostate and breast cancer). We demonstrate that chemical as well as transgenic suppression of APE1 impaired growth/colony formation and increased cytotoxicity of chemotherapeutic agent, whereas inhibited spontaneous as well as chemotherapy-induced DNA breaks, homologous recombination (HR) activity and genomic instability in all cancer cell types tested. Treatment with APE1 inhibitor also impaired tumor growth and significantly increased efficacy of a chemotherapeutic agent in a subcutaneous mouse model of EAC. Overexpression of APE1 in normal esophageal epithelial cells increased DNA breaks and HR activity, leading to massive mutational, copy number as well as karyotypic instability. Evaluation of by whole genome sequencing identified HR as the top mutational process activated by APE1. Normal cells overexpressing APE1 grew as tumors in mice and tumors removed from mice displayed additional karyotypic changes, providing evidence of genomic instability in vivo. Overall, our data demonstrate that elevated APE1 dysregulates HR activity, G2/M checkpoint and genome stability thus contributing to tumorigenesis and chemoresistance in cancer. Therefore, inhibitors of APE1 have potential to inhibit growth and increase cytotoxicity of chemotherapeutic agents while minimizing spontaneous as well as chemotherapy-induced genomic damage and instability in EAC and other solid tumors.

cancer biology↗
bioRxiv2022

Two complete genomes of male-killing Wolbachia infecting Ostrinia moth species illuminate their evolutionary dynamics and association with hosts

Wolbachia is an extremely widespread endocellular symbiont which causes reproductive manipulation on various arthropod hosts. Male progenies are killed in Wolbachia-infected lineages of the Japanese Ostrinia moth population. While the mechanism of male killing and the evolutionary interaction between host and symbiont are significant concerns for this system, the absence of Wolbachia genomic information has limited approaches to these issues. We determined the complete genome sequences of wFur and wSca, the male-killing Wolbachia of O. furnacalis and O. scapulalis. The two genomes shared an extremely high degree of homology, with over 95% of the predicted protein sequences being identical. A comparison of these two genomes revealed nearly minimal genome evolution, with a strong emphasis on the frequent genome rearrangements and the rapid evolution of ankyrin repeat-containing proteins. Additionally, we determined the mitochondrial genomes of both species infected lineages and performed phylogenetic analyses to deduce the evolutionary dynamics of Wolbachia infection in the Ostrinia clade. According to the inferred phylogenetic relationship, Wolbachia infection was established in the Ostrinia clade prior to the speciation of related species such as O. furnacalis and O. scapulalis. Simultaneously, the relatively high homology of mitochondrial genomes suggested recent Wolbachia introgression between infected Ostrinia species. The findings of this study collectively shed light on the host-symbiont interaction from an evolutionary standpoint. SignificanceDespite the growing number of publicly available Wolbachia genome sequences, only a few high-quality male-killer genomes exist, particularly those found in lepidopteran hosts. The complete genomes of two male-killing Wolbachia of Ostrinia moth hosts were determined in this study. The genomic data obtained here will be used to elucidate the mechanism of reproductive manipulation and the origins of this endosymbionts extraordinary diversity. Additionally, phylogenetic analysis of mitochondria and Wolbachia revealed the evolutionary history of Ostrinia hosts and Wolbachia. The inferred dynamic pattern of infection adds to our understanding of evolution and ecology of Wolbachia endosymbiont, a promising agent for biological pest control.

microbiology↗
bioRxiv2022

Genome size variation between pelagic and benthic communities across prokaryotic taxonomy and environmental gradients in the Baltic Sea

While theories and models have appeared to explain genome size as a result of evolutionary processes, little work has shown that genome sizes carry ecological signatures. Our work delves into the ecological implications of microbial genome size variation in benthic and pelagic habitats across environmental gradients of the brackish Baltic Sea. While depth is significantly associated with genome size in benthic and pelagic brackish metagenomes, salinity is only correlated to genome size in benthic metagenomes. Overall, we confirm that prokaryotic genome sizes in Baltic sediments (3.47 Mbp) are significantly bigger than in the water column (2.96 Mbp). While benthic genomes have a higher number of functions than pelagic genomes, the smallest genomes coded for a higher number of module steps per Mbp for most of the functions irrespective of their environment. Some examples of this functions are amino acid metabolism and central carbohydrate metabolism. However, we observed that nitrogen metabolism was almost absent in pelagic genomes and was mostly present in benthic genomes. Finally, we also show that Bacteria inhabiting Baltic sediments and water column not only differ in taxonomy, but also in their metabolic potential, such as the Wood-Ljungdahl pathway or the presence of different hydrogenases. Our work shows how microbial genome size is linked to abiotic factors in the environment, metabolic potential and taxonomic identity of Bacteria and Archaea within aquatic ecosystems.

microbiology↗
bioRxiv2022

PlantTribes2: tools for comparative gene family analysis in plant genomics

Plant genome-scale resources are being generated at an increasing rate as sequencing technologies continue to improve and raw data costs continue to fall; however, the cost of downstream analyses remains large. This has resulted in a considerable range of genome assembly and annotation qualities across plant genomes due to their varying sizes, complexity, and the technology used for the assembly and annotation. To effectively work across genomes, researchers increasingly rely on comparative genomic approaches that integrate across plant community resources and data types. Such efforts have aided the genome annotation process and yielded novel insights into the evolutionary history of genomes and gene families, including complex non-model organisms. The essential tools to achieve these insights rely on gene family analysis at a genome-scale, but they are not well integrated for rapid analysis of new data, and the learning curve can be steep. Here we present PlantTribes2, a scalable, easily accessible, highly customizable, and broadly applicable gene family analysis framework with multiple entry points including user provided data. It uses objective classifications of annotated protein sequences from existing, high-quality plant genomes for comparative and evolutionary studies. PlantTribes2 can improve transcript models and then sort them, either genome-scale annotations or individual gene coding sequences, into pre-computed orthologous gene family clusters with rich functional annotation information. Then, for gene families of interest, PlantTribes2 performs downstream analyses and customizable visualizations including, (1) multiple sequence alignment, (2) gene family phylogeny, (3) estimation of synonymous and non-synonymous substitution rates among homologous sequences, and (4) inference of large-scale duplication events. We give examples of PlantTribes2 applications in functional genomic studies of economically important plant families, namely transcriptomics in the weedy Orobanchaceae and a core orthogroup analysis (CROG) in Rosaceae. PlantTribes2 is freely available for use within the main public Galaxy instance and can be downloaded from GitHub or Bioconda. Importantly, PlantTribes2 can be readily adapted for use with genomic and transcriptomic data from any kind of organism.

bioinformatics↗
bioRxiv2022

Reference genome sequences of Mythimna separata (Lepidoptera: Noctuidae)

Lepidopteran insects are an important group of animals, among which some are used as biochemical and physiological model species in the insect and silk industries, whereas others are major agricultural pests. Therefore, genome sequences of several lepidopteran insects have been reported thus far. The oriental armyworm, Mythimna separata, is an agricultural pest commonly used to study insect immune reactions and interactions with parasitoid wasps as hosts. To improve our understanding of these research topics, reference genome sequences were constructed in the present study. Using long-read and short-read sequence data, de novo assembly and polishing were performed, and haplotigs were purged. Subsequently, gene predictions and functional annotations were performed. To search for orthologs of the Toll and immune deficiency (IMD) pathways and C-type lectins, annotation data analysis, BLASTp, and Hummer scans were performed. The M. separata genome is 682 Mbp; its contig N50 was 2.7 Mbp with 21,970 genes and 24,452 coding sites predicted. All orthologs of the core components of the Toll and IMD pathways and 105 C-type lectins were identified. These results suggest that the genome data were of sufficient quality as reference genome data and could contribute to promoting M. separata and lepidopteran research at the molecular and genome levels. Simple SummaryThe oriental armyworm, Mythimna separata, an agricultural pest, is commonly used to study insect immune reactions and interactions with parasitoid wasps. To promote such studies, a reference genome was constructed. The M. separata genome is 682Mbp long--a size comparable to that of other lepidopteran insects. The contig N50 value of the genome is 2.7 Mb, which indicates sufficient quality to be used as reference genome data. Gene set data were constructed using genome and RNA-sequencing data; a total of 21,970 genes and 24,452 coding sites were predicted. Functional gene annotation was performed using the predicted amino acid sequences and reference gene set data of the model organism and other insect species as well as Unigene and Pfam datasets. Consequently, 45-80% of the amino acid sequences were annotated using these data sets. Using these data, most of the orthologs of core components in the Toll and immune deficiency (IMD) pathways were identified, suggesting the presence of these two pathways in M. separata. Additionally, 105 C-type lectins were identified in the M. separata genome, which were more numerous than those in other insect species, suggesting that these genes may be duplicated.

bioinformatics↗
bioRxiv2023

Novel genomic offset metrics account for local adaptation in climate suitability forecasts and inform assisted migration

Local adaptation is increasingly being integrated into macroecological models, offering an evolutionary perspective that has been largely missing from climate change biogeography. Genomic offsets, which quantify the disruption of existing genotype-environment associations under environmental change, are an informative landscape genomic tool that allows for the incorporation of intra-specific adaptive differentiation into forecasts of climate suitability and thus management planning. Gradient forest (GF), a method originally developed to model community turnover along environmental gradients, is now the most commonly used approach in genomic offset estimation. However, major hurdles in the application of GF-derived offsets are (1) an inability to interpret the absolute magnitude of genomic offsets in a biologically meaningful way and (2) uncertainty in how genomic offsets compare to established species-level approaches like Ecological Niche Models (ENMs). We used both ENMs and novel, genomic offset metrics derived from GF modeling of genomic variation along climatic gradients to assess the climate change vulnerability of red spruce (Picea rubens), a cool-temperate tree species endemic to eastern North America. We show how genomic offsets can be standardized relative to contemporary genomic variation across the landscape to better represent their biological significance and facilitate comparisons among studies. In three common gardens, we found a significant negative relationship between standardized genomic offsets and red spruce growth and higher explanatory power for standardized offsets than (raw) climate transfer distances. We also derived new threshold-based metrics that we term Donor and Recipient Importance and which quantify transferability of propagules between donor and recipient localities while minimizing disruption of genotype-environment associations. This approach leverages landscape genomic information to account for local adaptation when predicting climate suitability. ENMs and our novel genomic offset metrics largely agreed in forecasting drastic northward range shifts. Combining several offset-based metrics, we show that the projected northward shift of suitable climate mainly applies to populations located in the center and northern parts of the current range, whereas southern populations might be able to persist in situ owing to specific local climate adaptations. The novel metrics thus yield refined, region-specific prognoses for local persistence and show how management could be improved by considering assisted migration.

ecology↗
bioRxiv2023

Mitochondrial Genome Diversity across the Subphylum Saccharomycotina

Eukaryotic life depends on the functional elements encoded by both the nuclear genome and organellar genomes, such as those contained within the mitochondria. The content, size, and structure of the mitochondrial genome varies across organisms with potentially large implications for phenotypic variance and resulting evolutionary trajectories. Among yeasts in the subphylum Saccharomycotina, extensive differences have been observed in various species relative to the model yeast Saccharomyces cerevisiae, but mitochondrial genome sampling across many groups has been scarce, even as hundreds of nuclear genomes have become available. By extracting mitochondrial assemblies from existing short-read genome sequence datasets, we have greatly expanded both the number of available genomes and the coverage across sparsely sampled clades. Comparison of 353 yeast mitochondrial genomes revealed that, while size and GC content were fairly consistent across species, those in the genera Metschnikowia and Saccharomyces trended larger, while several species in the order Saccharomycetales, which includes S. cerevisiae, exhibited lower GC content. Extreme examples for both size and GC content were scattered throughout the subphylum. All mitochondrial genomes shared a core set of protein-coding genes for Complexes III, IV, and V, but they varied in the presence or absence of mitochondrially-encoded canonical Complex I genes. We traced the loss of Complex I genes to a major event in the ancestor of the orders Saccharomycetales and Saccharomycodales, but we also observed several independent losses in the orders Phaffomycetales, Pichiales, and Dipodascales. In contrast to prior hypotheses based on smaller-scale datasets, comparison of evolutionary rates in protein-coding genes showed no bias towards elevated rates among aerobically fermenting (Crabtree/Warburg-positive) yeasts. Mitochondrial introns were widely distributed, but they were highly enriched in some groups. The majority of mitochondrial introns were poorly conserved within groups, but several were shared within groups, between groups, and even across taxonomic orders, which is consistent with horizontal gene transfer, likely involving homing endonucleases acting as selfish elements. As the number of available fungal nuclear genomes continues to expand, the methods described here to retrieve mitochondrial genome sequences from these datasets will prove invaluable to ensuring that studies of fungal mitochondrial genomes keep pace with their nuclear counterparts.

genetics↗
bioRxiv2023

PoMeLo: a systematic computational approach to predicting metabolic loss in pathogen genomes

BackgroundGenome streamlining, the process by which genomes become smaller and encode fewer genes over time, is a common phenomenon among pathogenic bacteria. This reduction is driven by selection for faster replication and minimized energy expenditure in a nutrientrich environment. As pathogens evolve to become more reliant on the host, metabolic genes and resulting capabilities are lost in favor of siphoning metabolites from the host. Characterizing genome streamlining, gene loss, and pathway degradation can be useful in assessing pathogen metabolic dependency on host metabolism and identifying potential targets for host-directed therapeutics. ResultsPoMeLo (Predictor of Metabolic Loss) is a novel evolutionary genomics-guided computational approach for identifying metabolic gaps in the genomes of pathogenic bacteria. PoMeLo leverages a centralized public database of high-quality genomes and annotations and allows the user to compare an unlimited number of genomes across individual genes and pathways. PoMeLo runs locally using user-friendly prompts in a matter of minutes and generates tabular and visual outputs for users to compare predicted metabolic capacity between groups of bacteria and individual species. Each pathway is assigned a Predicted Metabolic Loss (PML) score to assess the magnitude of genome streamlining. Optionally, PoMeLo places results in an evolutionary context by including phylogenetic relationships in visual outputs. It can also initially compute phylogenetically-weighted mean genome sizes to identify genome streamlining events. Here we describe PoMeLo and demonstrate its use in identifying metabolic gaps in genomes of pathogenic Treponema species. ConclusionsPoMeLo represents an advance over existing methods for identifying metabolic gaps in genomic data, allowing comparison across large numbers of genomes and placing the resulting data in a phylogenetic context. PoMeLo is freely available for academic and nonacademic use at https://github.com/czbiohub-sf/pomelo.

bioinformatics↗
bioRxiv2023

Geo-genomic predictors of genetree heterogeneity in an Amazonian bird (Thamnophilus aethiops)

Can knowledge about genome architecture inform biogeographic and phylogenetic inference? Selection, drift, recombination, and gene flow interact to produce a genomic landscape of divergence wherein patterns of differentiation and genealogy vary nonrandomly across the genomes of diverging populations. For instance, genealogical patterns that arise due to gene flow should be more likely to occur on smaller chromosomes, which experience high recombination, whereas those tracking histories of geographic isolation (reduced gene flow caused by a barrier) and divergence should be more likely to occur on larger and sex chromosomes. In Amazonia, populations of many bird species diverge and introgress across rivers, resulting in reticulated genomic signals. Herein, we used reduced representation genomic data to disentangle the evolutionary history of four populations of an Amazonian antbird, Thamnophilus aethiops, whose biogeographic history was associated with the dynamic evolution of the Madeira River Basin. Specifically, we evaluate whether a large river capture event ca. 200 kya, gave rise to reticulated genealogies in the genome by making spatially explicit predictions about isolation and gene flow based on knowledge about genomic processes. We first estimated chromosome-level phylogenies and recovered two primary topologies across the genome. The first topology (T1) was most consistent with predictions about population divergence, and was recovered for the Z chromosome. The second (T2), was consistent with predictions about gene flow upon secondary contact. To evaluate support for these topologies, we trained a convolutional neural network to classify our data into alternative diversification models and estimate demographic parameters. The best-fit model was concordant with T1 and included gene flow between non-sister taxa. Finally, we modeled levels of divergence and introgression as functions of chromosome length, and found that smaller chromosomes experienced higher gene flow. Given that (1) gene-trees supporting T2 were more likely to occur on smaller chromosomes and (2) we found lower levels of introgression on larger chromosomes (and especially the Z-chromosome), we argue that T1 represents the history of population divergence across rivers and T2 the history of secondary contact due to barrier loss. Our results suggest that a significant portion of genomic heterogeneity arises due to extrinsic biogeographic processes such as river capture interacting with intrinsic processes associated with genome architecture. Future biogeographic studies would benefit from accounting for genomic processes, as different parts of the genome reveal contrasting, albeit complementary histories, all of which are relevant for disentangling the intricate geogenomic mechanisms of biotic diversification.

evolutionary biology↗
bioRxiv2023

Diversity for commonality in the evolutionary changes of the reduced genome to regain the growth fitness

As the genome encodes the information crucial for cell growth, a sizeable genomic deficiency often causes a significant decrease in growth fitness. Whether and how the decreased growth fitness caused by genome reduction could be compensated by evolution was investigated here. Experimental evolution with an Escherichia coli strain carrying a reduced genome was conducted in multiple lineages for approximately 1,000 generations. The growth rate, which was largely declined due to genome reduction, was considerably recovered, associated with the improved carrying capacity. Genome mutations accumulated during evolution were significantly varied across the evolutionary lineages and were randomly localized on the reduced genome. Transcriptome reorganization showed a common evolutionary direction and conserved the chromosomal periodicity, regardless of highly diversified gene categories, regulons, and pathways enriched in the differentially expressed genes. Genome mutations and transcriptome reorganization caused by evolution, which were found to be dissimilar to those caused by genome reduction, must have followed divergent mechanisms in individual evolutionary lineages. Gene network reconstruction successfully identified three gene modules functionally differentiated, which were responsible for the evolutionary changes of the reduced genome in growth fitness, genome mutation, and gene expression, respectively. The diversity in evolutionary approaches improved the growth fitness associated with the homeostatic transcriptome architecture as if the evolutionary compensation for genome reduction was like all roads leading to Rome.

evolutionary biology↗
bioRxiv2023

High-sensitivity whole-genome recovery of single viral species in environmental samples

Characterizing unknown viruses is essential for understanding viral ecology and preparing against viral outbreaks. Recovering complete genome sequences from environmental samples remains computationally challenging using metagenomics, especially for low-abundance species with uneven coverage. This work presents a method for reliably recovering complete viral genomes from complex environmental samples. Individual genomes are encapsulated into droplets and amplified using multiple displacement amplification. A novel gene detection assay, which employs an RNA-based probe and an exonuclease, selectively identifies droplets containing the target viral genome. Labeled droplets are sorted using a microfluidic sorter, and genomes are extracted for sequencing. Validation experiments using a sewage sample spiked with two known viruses demonstrate the methods efficacy. We achieve 100% recovery of the spiked-in SV40 (Simian virus 40, 5243bp) genome sequence with uniform coverage distribution, and approximately 99.4% for the larger HAd5 genome (Human Adenovirus 5, 35938bp). Notably, genome recovery is achieved with as few as one sorted droplet, which enables the recovery of any desired genomes in complex environmental samples, regardless of their abundance. This method enables targeted characterizations of rare viral species and whole-genome amplification of single genomes for accessing the mutational profile in single virus genomes, contributing to an improved understanding of viral ecology.

bioengineering↗
bioRxiv2023

CpG Island Definition and Methylation Mapping of the T2T-YAO Genome

Precisely defining and mapping all cytosine positions and their clusters, known as CpG islands (CGIs), as well as their methylation status are pivotal for genome-wide epigenetic studies, especially when population-centric reference genomes are ready for timely application. Here we first align the two high-quality reference genomes, T2T-YAO and T2T-CHM13, from different ethnic backgrounds in a base-by-base fashion and compute their genome-wide density-defined and position-defined CGIs. Second, mapping some representative genome-wide methylation data from selected organs onto the two genomes, we find that there are about 4.7-5.8% sequence divergency of variable categories depending on quality cutoffs. Genes among the divergent sequences are mostly associated with neurological functions. Moreover, CGIs associated with the divergent sequences are significantly different with respect to CpG density and observed CpG/expected CpG (O/E) ratio between the two genomes. Finally, we find that the T2T-YAO genome not only has a greater CpG site coverage than that of the T2T-CHM13 genome when whole-genome bisulfite sequencing (WGBS) data from the European and American populations are mapped to each reference, but also show more hyper-methylated CpG sites as compared to the T2T-CHM13 genome. Our study suggests that future genome-wide epigenetic studies of the Chinese populations rely on both acquisition of high-quality methylation data and subsequent precision CGI mapping based on the Chinese T2T reference.

bioinformatics↗
bioRxiv2023

Hybracter: Enabling Scalable, Automated, Complete and Accurate Bacterial Genome Assemblies

Improvements in the accuracy and availability of long-read sequencing mean that complete bacterial genomes are now routinely reconstructed using hybrid (i.e. short- and long-reads) assembly approaches. Complete genomes allow a deeper understanding of bacterial evolution and genomic variation beyond single nucleotide variants (SNVs). They are also crucial for identifying plasmids, which often carry medically significant antimicrobial resistance (AMR) genes. However, small plasmids are often missed or misassembled by long-read assembly algorithms. Here, we present Hybracter which allows for the fast, automatic, and scalable recovery of near-perfect complete bacterial genomes using a long-read first assembly approach. Hybracter can be run either as a hybrid assembler or as a long-read only assembler. We compared Hybracter to existing automated hybrid and long-read only assembly tools using a diverse panel of samples of varying levels of long-read accuracy with manually curated ground truth reference genomes. We demonstrate that Hybracter as a hybrid assembler is more accurate and faster than the existing gold standard automated hybrid assembler Unicycler. We also show that Hybracter with long-reads only is the most accurate long-read only assembler and is comparable to hybrid methods in accurately recovering small plasmids. Data SummaryO_LIHybracter is developed using Python and Snakemake as a command-line software tool for Linux and MacOS systems. C_LIO_LIHybracter is freely available under an MIT License on GitHub (https://github.com/gbouras13/hybracter) and the documentation is available at Read the Docs (https://hybracter.readthedocs.io/en/latest/). C_LIO_LIHybracter is available to install via PyPI (https://pypi.org/project/hybracter/) and Bioconda (https://anaconda.org/bioconda/hybracter). A Docker/Singularity container is also available at https://quay.io/repository/gbouras13/hybracter. C_LIO_LIAll code used to benchmark Hybracter, including the reference genomes, is publicly available on GitHub (https://github.com/gbouras13/hybracter_benchmarking) with released DOI (https://zenodo.org/doi/10.5281/zenodo.10910108) available at Zenodo. C_LIO_LIThe subsampled FASTQ files used for benchmarking are publicly available at Zenodo with DOI (https://doi.org/10.5281/zenodo.10906937). C_LIO_LIAll super accuracy simplex ATCC FASTQ reads sequenced as a part of this study can be found under BioProject PRJNA1042815. C_LIO_LIAll Hall et al. fast accuracy simplex and super accuracy duplex ATCC FASTQ read files (prior to subsampling) can be found in the SRA under BioProject PRJNA1087001. C_LIO_LIAll raw Lermaniaux et al. FASTQ read files and genomes (prior to subsampling) can be found in the SRA under BioProject PRJNA1020811. C_LIO_LIAll Staphylococcus aureus JKD6159 FASTQ read files and genomes can be found under BioProject PRJNA50759. C_LIO_LIAll Mycobacterium tuberculosis H37R2 FASTQ read files and genomes can be found under BioProject PRJNA836783. C_LIO_LIThe complete list of BioSample accession numbers for each benchmarked sample can be found in Supplementary Table 1. C_LIO_LIThe benchmarking assembly output files are publicly available on Zenodo with DOI (https://doi.org/10.5281/zenodo.10906937). C_LIO_LIAll Pypolca benchmarking outputs and code are publicly available on Zenodo with DOI (https://zenodo.org/doi/10.5281/zenodo.10072192). C_LI Impact StatementComplete bacterial genome assembly using hybrid sequencing is a routine and vital part of bacterial genomics, especially for identification of mobile genetic elements and plasmids. As sequencing becomes cheaper, easier to access and more accurate, automated assembly methods are crucial. With Hybracter, we present a new long-read first automated assembly tool that is faster and more accurate than the widely-used Unicycler. Hybracter can be used both as a hybrid assembler and with long-reads only. Additionally, it solves the problems of long-read assemblers struggling with small plasmids, with plasmid recovery from long-reads only performing on par with hybrid methods. Hybracter can natively exploit the parallelisation of high-performance computing (HPC) clusters and cloud-based environments, enabling users to assemble hundreds or thousands of genomes with one line of code. Hybracter is available freely as source code on GitHub, via Bioconda or PyPi.

bioinformatics↗
bioRxiv2024

A neutral process of genome reduction in marine bacterioplankton

Marine bacterioplankton communities are dominated by cells equipped with small genomes. Streamlining selection has been accepted as the main force driving their genome reduction. Here, we report that a neutral evolutionary mechanism governs genome reduction in the Roseobacter group that represents 5-20% of the bacterioplankton cells in coastal waters. Using representative strains that fall into three genome size groups (2-3, 3-4, and 4-5 Mbp), we measured their genomic mutation rates () through long-term mutation accumulation experiments followed by genome sequencing the resulting 437 mutant lines. We further calculated their effective population sizes (Ne) based on and the neutral genetic diversity of the studied species, the latter estimated based on multiple genome sequences of natural isolates collected from global oceans with their population structure considered. A surprising finding is that Ne scales positively with genome size, which is the opposite of the expectation from the streamlining selection theory. As the strength of random genetic drift is the inverse of Ne, this result instead suggests drift as the primary driver of genome reduction. Additionally, we report a negative scaling between and genome size, which is the first experimental evidence for the long-lasting hypothesis that mutation rate increases play a part in marine bacterial genome reduction. As scales inversely with Ne, genetic drift appears to be the ultimate cause of genome reduction in these Roseobacters. Our finding discounts, but is insufficient to reject, the streamlining theory because streamlining process is expected to be more effective in oligotrophic open ocean waters.

microbiology↗