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bioRxiv2024

Recovery of highly contiguous genomes from complex terrestrial habitats reveals over 15,000 novel prokaryotic species and expands characterization of soil and sediment microbial communities

Genomes are fundamental to understanding microbial ecology and evolution. The emergence of high-throughput, long-read DNA sequencing has enabled recovery of microbial genomes from environmental samples at scale. However, expanding the microbial genome catalogue of soils and sediments has been challenging due to the enormous complexity of these environments. Here, we performed deep, long-read Nanopore sequencing of 154 soil and sediment samples collected across Denmark and through an optimised bioinformatics pipeline, we recovered genomes of 15,314 novel microbial species, including 4,757 high-quality genomes. The recovered microbial genomes span 1,086 novel genera and provide the first high-quality reference genomes for 612 previously known genera, expanding the phylogenetic diversity of the prokaryotic tree of life by 8 %. The long-read assemblies also enabled the recovery of thousands of complete rRNA operons, biosynthetic gene clusters and CRISPR-Cas systems, all of which were underrepresented and highly fragmented in previous terrestrial genome catalogues. Furthermore, the incorporation of the recovered MAGs into public genome databases significantly improved species-level classification rates for soil and sediment metagenomic datasets, thereby enhancing terrestrial microbiome characterization. With this study, we demonstrate that long-read sequencing and optimised bioinformatics, allows cost-effective recovery of high-quality microbial genomes from highly complex ecosystems, which remain the largest untapped source of biodiversity for expanding genome databases and filling in the gaps of the tree of life.

bioinformatics↗

bioRxiv2024

Genomic Characterisation of Group B Streptococcus from Argentina: Insights into Prophage Diversity, Virulence Factors and Antibiotic Resistance Genes

Group B Streptococcus (GBS) is a commensal bacterium that can cause severe infections in infants and adults with comorbidities. Resistance and reduced susceptibility to antibiotics is continually on the rise, and vaccines remain in-development. Prophages have been reported to contribute to GBS evolution and pathogenicity. However, no studies are available to date on prophage contribution to the epidemiology of GBS isolates from humans in South America. In the context of an Argentinian multicentric study, we had previously phenotypically characterised 365 human GBS isolates from invasive disease, urinary infections and maternal colonisation. These isolates had been whole genome sequenced and their prophage presence bioinformatically determined. In this study, we genomically characterised the isolates and analysed the prophage content in the context of the epidemiological data. The phylogenetic analysis of the 365 genomes with 103 GBS from public databases revealed that Argentinian GBS were related to isolates from around the world. The most prevalent lineages, independently of the isolate source, were CC23/Ia and CC12/Ib. Genes encoding virulence factors involved in immune response evasion, tissue damage and adherence to host tissues and invasion were found in all of the genomes in accordance with previously described lineage distribution. According to the prevalent capsular types and the distribution of specific virulence factors in Argentinian GBS, over 95% coverage would be expected from the vaccines currently under development. Antibiotic resistance determinants (ARDs) to at least one antibiotic class were found in 90% of the genomes, including novel mutations in pbp2x, while more than 15% carried ARDs to 3 or more classes. GBS collected from urinary infections carried a significantly higher proportion of ARDs to multiple antibiotic classes than the rest of the isolates. A total of 454 prophages were found among the 468 genomes analysed, which were classified into 23 prophage types. Prophage presence exhibited variations based on GBS clonal complex and capsular type. A possible association between an increased GBS pathogenicity and the carriage of prophages with integrase type GBSInt8 and/or the presence of genes that encode the Phox Homology domain has been observed. The highest prevalence of prophages per genome was found in lineages CC17/III and CC19/III, while the lowest amount was observed in CC12/Ib. Overall, the highest density of prophages, virulence factors and ARDs determinants was found in CC19 isolates, mostly of capsular type III, independently of the isolates source. This is the first analysis of the human-associated GBS population in South America based on whole genome sequencing data, which will make a significant contribution to future studies on the global GBS population structure. Data summarySupplementary results and figures can be found in Supplementary Material 1. Supplementary tables can be found in Supplementary Material 2. All datasets analysed in this study are detailed in the Supplementary Materials. Metadata about the genomes analysed can be found in the microreact project created for this study: https://microreact.org/project/gbs-pangenomic-analysis. Impact statementIn Latin America studies on the epidemiology of Group B Streptococcus (GBS) are scarce, especially those describing clonal complex and serotype distribution, and the role of prophages in GBS epidemiology has not been studied. This article addresses the first genomic characterisation of the human-isolated GBS population in Latin America based on whole genome sequencing data, with special focus on the analysis of prophage content. We determine the clonal complex and serotype distribution of 365 GBS isolates collected from clinical samples in an Argentinian multicenter study and analyse the presence of prophages and virulence and antibiotic resistance determinants in the context of the epidemiological data. Through these analyses, we were able to determine how GBS population structure in Argentina differs from other parts of the world and to predict the potential coverage of the in-development GBS vaccines. We also found a possible association between the carriage of certain types of prophages and an increased GBS pathogenicity. In the context of increased global efforts to develop new strategies to prevent GBS infections through vaccine development, this study makes a significant contribution to our understanding of the global GBS population structure.

bioinformatics↗

bioRxiv2025

Unsupervised Whole-Genome Representation Learning Captures Bacterial Phenotypes

Shifting from hand-crafted to learned representations of data has revolutionized fields like natural language processing and computer vision. Despite this, current approaches to bacterial phenotype prediction from the genome rely on training machine learning models on hand-crafted features, often binary indicators or counts of the presence of different conserved genomic elements and protein domains. Defining these shared elements and domains as our "genomic element vocabulary", we tokenize entire bacterial genomes as sequences of these conserved elements and take advantage of advances in long-context language modeling to perform self-supervised whole-genome representation learning (WGRL). Through multi-task pretraining on a phylogenetically diverse dataset of hundreds of thousands of bacterial genomes, we present a genomic language model which produces representations of input genomes with features predictive of a broad range of phenotypes. We assess the quality of the learned representations through k-nearest neighbours prediction of 25 bacterial phenotypes, finding our WGRL representations more predictive than standard protein domain presence/absence representations for 23/25 different phenotypes. We additionally find the WGRL representations are robust to both poor genome assembly quality and incompleteness. Through learning the relationships between evolutionarily conserved genomic elements with self-supervised long-context language modeling, we demonstrate the first approach for extracting general-purpose whole-genome representations while preserving gene order.

bioinformatics↗

bioRxiv2025

Evolutionary Constraints on RNA Polymerase Gene Positioning in the Genome of Fast-Growing Bacteria..

How gene order along chromosomes affects cellular homeostasis and genome evolution remains poorly understood. Bacterial chromosomes are organized along the replication origin (oriC)-terminus (ter) axis. The spatial arrengement of genes within this axis may influence cellular physiology, genome evolution, and transcriptional regulation. The catalytic core of the sole bacterial RNA polymerase (RNAP) is encoded by rpoB and rpoC within the universally conserved rplKAJL-rpoBC locus. In fast growing bacteria this locus is located near oriC potentially linking genome organization to cellular physiology. Here, we investigated the functional relevance of rplKAJL-rpoBC chromosomal positioning by relocating it within Vibrio cholerae genome. While viable, strains harboring rplKAJL-rpoBC far from oriC exhibited nutrient-dependent fitness defects. These phenotypes correlated with reduced locus copy number and RNAP abundance, without changes in its subcellular distribution. Introducing a second copy of rpIKAJL-rpoBC at a distal genomic location from oriC abolished the phenotypes, demonstrating that replication-associated gene dosage effects were the main mechanism behind the observed physiological changes. Further uncoupling rpoB and rpoC from the rplKAJL-rpoBC locus revealed that fitness costs were specifically linked to RNAP gene relocation. Our results reveal that selective pressures act to maintain RNAP-encoding genes near oriC to optimize replication-associated dosage effects, ensuring efficient RNAP production during exponential growth. We discuss the relationship between gene order and ecological strategies linked to bacterial growth. Our work underscores gene order as an underestimated but critical factor shaping cellular physiology and genome evolution. SignificanceBacterial chromosomes have a highly plastic gene content but the importance of their spatial organization and gene order is started to be appreciated as a driver of cellular physiology. Growing evidence suggests that the spatial organization of bacterial genomes plays a critical role in gene expression, replication dynamics, and fitness. In this study, we demonstrate that the conserved positioning of rpoB and rpoC, encoding the catalytic core of RNA polymerase (RNAP), near the replication origin (oriC) is essential for optimal bacterial growth. By relocating the rplKAJL-rpoBC locus in Vibrio cholerae, we show that its chromosomal position directly impacts nutrient-dependent fitness, RNAP abundance, and replication-associated gene dosage effects. These findings suggest that selective pressures maintain genes encoding RNAP near oriC to ensure efficient transcriptional output during exponential growth. More broadly, our study highlights gene order as an underappreciated factor in bacterial genome evolution, beyond its known roles in operon structure and transcriptional regulation. Given the fundamental role of RNAP, our results have implications for understanding bacterial ecological strategies, adaptation, and synthetic genome design. By integrating positional genomics with physiological studies, we provide a theoretical and experimental framework for investigating how genome architecture shapes cellular function.

microbiology↗

bioRxiv2025

Calicivirus assembly and stability are mediated by the N-terminal domain of the capsid protein with the involvement of the viral genome

Caliciviruses are important human and animal pathogens that cause varying clinical signs including gastroenteritis, respiratory illness, and hepatitis. Despite the availability of numerous calicivirus structures, relatively little is known about the mechanisms of capsid assembly and stability, or about genome packaging. Here we present the atomic structure of the RHDV virion and several related non-infectious virus-like particles, determined using cryo-EM at 2.5-3.3 [A] resolution. The inherent molecular switch, responsible for the conformational flexibility of the capsid protein VP1, is located in its N-terminal arm (NTA). The NTA establishes an extensive network of interactions on the inner capsid surface that stabilizes the hexamers and pentamers. For this structural polymorphism, we show that the NTA must interact with the RNA viral genome, that is, the genomic RNA acts concomitantly with the NTA as a molecular switch. The NTA-RNA interaction leads to specific conformational states that result in two types of VP1 dimers (the basic building blocks) necessary for T=3 capsid assembly. In addition, we used atomic force microscopy (AFM) to assess whether differences in genomic RNA content influence viral properties such as capsid stiffness in physiological conditions. These analyses highlight the mechanical role of packed RNA genome in RHDV virions, as the virion capsid pentamers are strengthened by interactions of the NTA star-like structure promoted by the viral genome. These results indicate that the interactions between the NTA and the viral genome guide the conformational states of VP1 dimers, directing capsid assembly and modulating its mechanical properties. Through interference with intermediate assemblies, the NTA network promoted by the genome could be an attractive target in future antiviral strategies. Author SummaryRabbit hemorrhagic disease virus (RHDV) is a highly contagious, often fatal calicivirus that poses a significant threat to rabbit populations worldwide. Although considerable progress has been made in elucidating the structural organization of RHDV capsids at near-atomic resolution, information regarding the nucleic acid organization within these virions and its interactions with the viral capsid remains limited. Understanding these interactions is essential for identifying mechanisms of structural polymorphism of the capsid protein, as well as virion assembly and stability. We report the cryo-electron microscopy structure, at near-atomic resolution, of RHDV virions and distinct virus-like particles, together with characterization of their biophysical properties using atomic force microscopy. The N-terminal region of the capsid protein, essential for its structural polymorphism, requires its interaction with genomic RNA. The interaction between N-terminal arms and the viral genome governs the conformational states of capsid protein dimers, thereby orchestrating capsid assembly and influencing its biophysical properties. By interfering with intermediate assemblies, the NTA network promoted by the genome could constitute an attractive target in antiviral strategies for several major human calicivirus pathogens that cause acute gastroenteritis and respiratory illness, including norovirus and sapovirus.

biophysics↗

bioRxiv2025

Leviathan: A fast, memory-efficient, and scalable taxonomic and pathway profiler for next generation sequencing (pan)genome-resolved metagenomics and metatranscriptomics

Functional profiling of meta-omics is essential for understanding microbial communities, yet support for custom genome-resolved reference databases is limited. We introduce Leviathan for integrated taxonomic and functional profiling at both genome and pangenome resolution. Leviathan combines Sylph for ultrafast alignment-free taxonomic profiling with Salmon for pseudo-alignment-based read quantification in DNA-space against (pan)genome-resolved gene catalogs, producing dual metrics per (pan)genome: pathway abundance and graph-based pathway coverage. Benchmarking alignment backends on synthetic metagenomes, we show that DNA-space pseudo-alignments retain competitive (pan)genome-level classification performance compared to traditional alignment, reducing computational runtime and memory requirements, while translated searches in protein-space lose classification resolution from ambiguous mapping events. Leviathans utility is demonstrated through two case studies: a marine plastisphere metagenomics dataset analyzing metabolic shifts between early and mature biofilm communities and a dental caries metatranscriptomics dataset where co-expression network analysis identified organism-specific transcriptional patterns diagnostic of health and disease states. Leviathan is available at https://github.com/jolespin/leviathan. ImportanceUnderstanding what microbes can do, not just which ones are present, is central to translating microbiome research into actionable insight. Existing functional profiling tools either rely on fixed reference databases or require complex multi-step pipelines when applied to custom genome collections, and none natively compute per-(pan)genome pathway abundance and graph-based pathway completeness in a single workflow. This limits the ability for researchers to directly compare functional profiles to tangential analyses on their specific genome catalogs. Leviathan addresses this gap with integrated taxonomic and functional profiling against user-defined (pan)genome-resolved references using pseudo-alignment, achieving competitive classification accuracy with substantially lower runtime and memory requirements compared to current methods. Native pangenome support enables routine quantification of metabolic potential and transcriptional activity at both genome and pangenome resolution, revealing functional variation across related strains that single-genome or community-level analyses obscure.

bioinformatics↗

bioRxiv2025

Genome size variation is attributed to adaptive purging of transposable elements

A substantial variation in genome size has been observed among individuals of the same species. Theory predicts that increased genome size may confer an advantage in populations with small effective size. However, contradictory evidence for the correlation with environmental variation, and limited understanding of the underlying genetic mechanisms, has cast doubts on the adaptive role of genome size variation. To address this, we studied two Hordeum species which were collected at the same sites along a wide range of environments but different in life habit (annual/perrenial) and mating strategy (self/out-crossing). We observed significant variation in genome size, with differences of up to 10% consistently detected in both species. Mating system influenced only the proportion of variation maintained within populations but not the overall range of genome size. In both species, drought emerged as the primary environmental factor associated with genome expansion, with transposable elements (TE) accumulation identified as the main driver of this expansion. Genome-wide association studies revealed that TE silencing is the key mechanism regulating genome size, and that selection favors smaller genomes among individuals growing at suitable habitats. Under increasingly stressful conditions, the regulation of TE activity fades, leading to TE accumulation and genome expansion, thereby increasing genetic variation available for selection. This integrative study provides a comprehensive view of how genome size is regulated in natural populations and highlights its exaptive role in inducing genetic variation under environmental stress.

evolutionary biology↗

bioRxiv2025

Chromosome-scale haplotype genome assemblies for the Australian mango 'Kensington Pride' 1 and a wild relative, Mangifera laurina, provide insights into anthracnose-resistance and volatile 2 compound biosynthesis genes

Mango (Mangifera indica) is one of the most popular fruits cultivated in tropical and subtropical regions of the world. The availability of reference genomes helps to identify the genetic basis of important traits. Here we report assembled high-quality chromosome-level genomes for the Australian mango cultivar Kensington Pride, and M. laurina; a wild relative, which shows resistance to anthracnose disease. PacBio HiFi sequencing with higher genome coverage enabled the assembly of both genomes with 100% completeness. Genome sizes of Kensington Pride and M. laurina were 367 Mb and 379 Mb, respectively, with all 20 chromosomes in both genomes having telomeres at both ends. K-mer analysis revealed that these genomes are highly heterozygous and significant structural variations were identified between Kensington Pride, M. laurina, and the recently published genome of the cultivar Irwin. Functional annotation identified key genes involved in carotenoid, anthocyanin, and terpenoid biosynthesis, responsible for fruit color and flavour in mango. Furthermore, the presence of a SNP in {beta}-1,3-glucanase 2 gene associated with anthracnose resistance was analyzed. Whole genome duplication analysis confirmed that mangoes have undergone two polyploidization events during their evolution. Analysis revealed a conserved pattern of colinear genes, although many colinear blocks were also identified on non-homologous chromosomes. Practitioner PointsO_LIPacBio HiFi sequencing and high coverage produced genomes for Kensington Pride mango and M. laurina with 100% completeness, identifying all the telomeres in the assembled chromosomes. C_LIO_LISignificant structural variations were identified between Kensington Pride, M. laurina, and the published Irwin genome. C_LIO_LIGenes linked in the biosynthesis of unique terpenoids were identified, and the structural differences in the annotated {beta}-1,3-glucanase 2 genes associated with anthracnose resistance provide a resource for gene expression analysis in susceptible and resistant cultivars. C_LI

plant biology↗

bioRxiv2018

A Fast Adaptive Algorithm for Computing Whole-Genome Homology Maps

MotivationWhole-genome alignment is an important problem in genomics for comparing different species, mapping draft assemblies to reference genomes, and identifying repeats. However, for large plant and animal genomes, this task remains compute and memory intensive.\n\nResultsWe introduce an approximate algorithm for computing local alignment boundaries between long DNA sequences. Given a minimum alignment length and an identity threshold, our algorithm computes the desired alignment boundaries and identity estimates using kmer-based statistics, and maintains sufficient probabilistic guarantees on the output sensitivity. Further, to prioritize higher scoring alignment intervals, we develop a plane-sweep based filtering technique which is theoretically optimal and practically efficient. Implementation of these ideas resulted in a fast and accurate assembly-to-genome and genome-to-genome mapper. As a result, we were able to map an error-corrected whole-genome NA12878 human assembly to the hg38 human reference genome in about one minute total execution time and < 4 GB memory using 8 CPU threads, achieving more than an order of magnitude improvement in both runtime and memory over competing methods. Recall accuracy of computed alignment boundaries was consistently found to be > 97% on multiple datasets. Finally, we performed a sensitive self-alignment of the human genome to compute all duplications of length [≥] 1 Kbp and [≥] 90% identity. The reported output achieves good recall and covers 5% more bases than the current UCSC genome browsers segmental duplication annotation.\n\nAvailabilityhttps://github.com/marbl/MashMap\n\nContactadam.phillippy@nih.gov, aluru@cc.gatech.edu

bioinformatics↗

bioRxiv2018

FusoPortal: An interactive repository of hybrid MinION sequenced Fusobacterium genomes improves gene identification and characterization

Here we present FusoPortal, an interactive repository of Fusobacterium genomes that were sequenced using a hybrid MinION long-read sequencing pipeline, followed by assembly and annotation using a diverse portfolio of predominantly open-source software. Significant efforts were made to provide genomic and bioinformatic data as downloadable files, including raw sequencing reads, genome maps, gene annotations, protein functional analysis and classifications, and a custom BLAST server for FusoPortal genomes. FusoPortal has been initiated with eight complete genomes, of which seven were previously only drafts that varied from 24-67 contigs. We showcase that genomes in FusoPortal provide accurate open reading frame annotations, and have corrected a number of large genes (>3 kb) that were previously misannotated due to contig boundaries. In summary, FusoPortal (http://fusoportal.org) is the first database of MinION sequenced and completely assembled Fusobacterium genomes, and this central Fusobacterium genomic and bioinformatic resource will aid the scientific community in developing a deeper understanding of how this human pathogen contributes to an array of diseases including periodontitis and colorectal cancer.\n\nImportanceIn this study, we report a hybrid MinION whole genome sequencing pipeline, and describe the genomic characteristics of the first eight strains deposited in the FusoPortal database. This collection of highly accurate and complete genomes drastically improves upon previous multi-contig assemblies by correcting and newly identifying a significant number of open reading frames. We believe this resource will result in the discovery of proteins and molecular mechanisms used by an oral pathogen, with the potential to further our understanding of how F. nucleatum contributes to a repertoire of diseases including periodontitis, pre-term birth, and colorectal cancer

microbiology↗

bioRxiv2018

Genome plasticity, a key factor of evolution in prokaryotes

In prokaryotic genomes, the number of genes that belong to distinct functional classes shows apparent universal scaling with the total number of genes [1-5] (Fig. 1). This scaling can be approximated with a power law, where the scaling power can be sublinear, near-linear or super-linear. Scaling laws are robust under various statistical tests [4], across different databases and for different gene classifications [1-5]. Several models aimed at explaining the observed scaling laws have been proposed, primarily, based on the specifics of the respective biological functions [1, 5-8]. However, a coherent theory to explain the emergence of scaling within the framework of population genetics is lacking. We employ a simple mathematical model for prokaryotic genome evolution [9] which, together with the analysis of 34 clusters of closely related microbial genomes [10], allows us to identify the underlying forces that dictate genome content evolution. In addition to the scaling of the number of genes in different functional classes, we explore gene contents divergence to characterize the evolutionary processes acting upon genomes [11]. We find that evolution of the gene content is dominated by two factors that are specific to a functional class, namely, selection landscape and genome plasticity. Selection landscape quantifies the fitness cost that is associated with deletion of a gene in a given functional class or the advantage of successful incorporation of an additional gene. Genome plasticity, that can be considered a measure of evolvability, reflects both the availability of the genes of a given functional class in the external gene pool that is accessible to the evolving microbial population, and the ability of microbial genomes to accommodate these genes. The selection landscape determines the gene loss rate, and genome plasticity is the principal determinant of the gene gain rate.\n\nO_FIG O_LINKSMALLFIG WIDTH=197 HEIGHT=200 SRC=\"FIGDIR/small/357400_fig1.gif\" ALT=\"Figure 1\">\nView larger version (39K):\norg.highwire.dtl.DTLVardef@6df3e2org.highwire.dtl.DTLVardef@a69e8dorg.highwire.dtl.DTLVardef@f36a80org.highwire.dtl.DTLVardef@d519c9_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Scaling laws for all functional classes of the COGs. The number of genes in a given COG category is plotted against the total number of genes. Each point represents one genome from the analyzed set of 1490 genomes. The scaling is fitted to a power law which is indicated by a solid red line. The fitted scaling exponent is indicated in parentheses.\n\nC_FIG

microbiology↗

bioRxiv2018

RT States: systematic annotation of the human genome using cell type-specific replication timing programs

The replication timing (RT) program has been linked to many key biological processes including cell fate commitment, 3D chromatin organization and transcription regulation. Significant technology progress now allows to characterize the RT program in the entire human genome in a high-throughput and high-resolution fashion. These experiments suggest that RT changes dynamically during development in coordination with gene activity. Since RT is such a fundamental biological process, we believe that an effective quantitative profile of the local RT program from a diverse set of cell types in various developmental stages and lineages can provide crucial biological insights for a genomic locus. In the present study, we explored recurrent and spatially coherent combinatorial profiles from 42 RT programs collected from multiple lineages at diverse differentiation states. We found that a Hidden Markov Model with 15 hidden states provide a good model to describe these genome-wide RT profiling data. Each of the hidden state represents a unique combination of RT profiles across different cell types which we refer to as \"RT states\". To understand the biological properties of these RT states, we inspected their relationship with chromatin states, gene expression, functional annotation and 3D chromosomal organization. We found that the newly defined RT states possess interesting genome-wide functional properties that add complementary information to the existing annotation of the human genome.\n\nAUTHOR SUMMARYThe replication timing (RT) program is an important cellular mechanism and has been linked to many key biological processes including cell fate commitment, 3D chromatin organization and transcription regulation. Significant technology progress now allows us to characterize the RT program in the entire human genome. Results from these experiments suggest that RT changes dynamically across different developmental stages. Since RT is such a fundamental biological process, we believe that the local RT program from a diverse set of cell types in various developmental stages can provide crucial biological insights for a genomic locus. In the present study, we explored combinatorial profiles from 42 RT programs collected from multiple lineages at diverse differentiation states. We developed a statistical model consist of 15 \"RT states\" to describe these genome-wide RT profiling data. To understand the biological properties of these RT states, we inspected the relationship between RT states and other types of functional annotations of the genome. We found that the newly defined RT states possess interesting genome-wide functional properties that add complementary information to the existing annotation of the human genome.

bioinformatics↗

bioRxiv2018

A novel core genome approach to enable prospective and dynamic monitoring of infectious outbreaks

Whole-genome sequencing is increasingly adopted in clinical settings to identify pathogen transmissions. Currently, such studies are performed largely retrospectively, but to be actionable they need to be carried out prospectively, in which samples are continuously added and compared to previous samples. To enable prospective pathogen comparison, genomic relatedness metrics based on single nucleotide differences must be consistent across time, efficient to compute and reliable for a large variety of samples. The choice of genomic regions to compare, i.e., the core genome, is critical to obtain a good metric.\n\nWe propose a novel core genome method that selects conserved sequences in the reference genome by comparing its k-mer content to that of publicly available genome assemblies. The conserved-sequence genome is sample set-independent, which enables prospective pathogen monitoring. Based on clinical data sets of 3436 S. aureus, 1362 K. pneumoniae and 348 E. faecium samples, we show that the conserved-sequence genome disambiguates same-patient samples better than a core genome consisting of conserved genes. The conserved-sequence genome confirms outbreak samples with high accuracy: in a set of 2335 S. aureus samples, it correctly identifies 44 out of 45 outbreak samples, whereas the conserved gene method confirms 38 out of 45 outbreak samples.

bioinformatics↗

bioRxiv2018

How to make a rodent giant: Genomic basis and tradeoffs of gigantism in the capybara, the world’s largest rodent

Gigantism is the result of one lineage within a clade evolving extremely large body size relative to its small-bodied ancestors, a phenomenon observed numerous times in animals. Theory predicts that the evolution of giants should be constrained by two tradeoffs. First, because body size is negatively correlated with population size, purifying selection is expected to be less efficient in species of large body size, leading to a genome-wide elevation of the ratio of non-synonymous to synonymous substitution rates (dN/dS) or mutation load. Second, gigantism is achieved through higher number of cells and higher rates of cell proliferation, thus increasing the likelihood of cancer. However, the incidence of cancer in gigantic animals is lower than the theoretical expectation, a phenomenon referred to as Petos Paradox. To explore the genetic basis of gigantism in rodents and uncover genomic signatures of gigantism-related tradeoffs, we sequenced the genome of the capybara, the worlds largest living rodent. We found that dN/dS is elevated genome wide in the capybara, relative to other rodents, implying a higher mutation load. Conversely, a genome-wide scan for adaptive protein evolution in the capybara highlighted several genes involved in growth regulation by the insulin/insulin-like growth factor signaling (IIS) pathway. Capybara-specific gene-family expansions included a putative novel anticancer adaptation that involves T cell-mediated tumor suppression, offering a potential resolution to Petos Paradox in this lineage. Gene interaction network analyses also revealed that size regulators function simultaneously as growth factors and oncogenes, creating an evolutionary conflict. Based on our findings, we hypothesize that gigantism in the capybara likely involved three evolutionary steps: 1) Increase in body size by cell proliferation through the ISS pathway, 2) coupled evolution of growth-regulatory and cancer-suppression mechanisms, possibly driven by intragenomic conflict, and 3) establishment of the T cell-mediated tumor suppression pathway as an anticancer adaptation. Interestingly, increased mutation load appears to be an inevitable outcome of an increase in body size.\n\nAuthor SummaryThe existence of gigantic animals presents an evolutionary puzzle. Larger animals have more cells and undergo exponentially more cell divisions, thus, they should have enormous rates of cancer. Moreover, large animals also have smaller populations making them vulnerable to extinction. So, how do gigantic animals such as elephants and blue whales protect themselves from cancer, and what are the consequences of evolving a large size on the genetic health of a species? To address these questions we sequenced the genome of the capybara, the worlds largest rodent, and performed comparative genomic analyses to identify the genes and pathways involved in growth regulation and cancer suppression. We found that the insulin-signaling pathway was involved in the evolution of gigantism in the capybara. We also found a putative novel anticancer mechanism mediated by the detection of tumors by T-cells, offering a potential solution to how capybaras mitigated the tradeoff imposed by cancer. Furthermore, we show that capybara genome harbors a higher proportion of slightly deleterious mutations relative to all other rodent genomes. Overall, this study provides insights at the genomic level into the evolution of a complex and extreme phenotype, and offers a detailed picture of how the evolution of a giant body size in the capybara has shaped its genome.

evolutionary biology↗

bioRxiv2018

Genome wide screen reveals a specific interaction between autosome and X that is essential for hybrid male sterility

Hybrid male progeny from interspecies cross are more prone to sterility or inviability than hybrid female progeny, and the male sterility and inviability often demonstrate a parent-of-origin asymmetry. However, the underlying mechanism of asymmetric sterility or inviability remains elusive. We previously established a genome-wide hybrid incompatibility (HI) landscape between Caenorhabditis briggsae and C. nigoni by phenotyping a large collection of C. nigoni strains each carrying a C. briggsae introgression. In this study, we investigate the genetic mechanism of asymmetric sterility and inviability in both hybrid male and female progeny between the two species. Specifically, we performed reciprocal crosses between C. briggsae and different C. nigoni strains that each carries a GFP-labeled C. briggsae genomic fragment referred to as introgression, and scored the HI phenotypes in the F1 progeny. The aggregated introgressions cover 94.6% of the C. briggsae genome, including 100% of the X chromosome. Surprisingly, we observed that two C. briggsae X fragments that produce C. nigoni male sterility as an introgression rescued hybrid F1 sterility in males fathered by C. briggsae, indicating that at least two separate X-autosome interactions are involved in the hybrid male sterility. In addition, we identified another two C. briggsae genomic intervals on the Chromosome II or IV, respectively, which can rescue the inviability, but not the sterility, of hybrid F1 males fathered by C. nigoni, suggesting the involvement of differential epistatic interactions in the asymmetric hybrid male fertility and inviability. Importantly, backcrossing of the rescued sterile males with C. nigoni led to isolation of a 1.1-Mb genomic interval that specifically interacts with an X-linked introgression, which is essential for hybrid male fertility. We further identified three C. briggsae genomic intervals on the Chromosome I, II and III, respectively that produce inviability in all F1 progeny dependent or independent of the parent-of-origin. Taken together, we identified multiple independent interacting loci that are responsible for asymmetric hybrid male and female sterility and inviability, which provides important insights into the asymmetric HI and lays a foundation for their molecular characterization.\n\nAuthor summaryIt is common that closely related species can mate with each other, but their hybrid progeny are often sterile or inviable, especially in the male progeny. The mechanism underlying the asymmetric sterility or inviability remains poorly understood. We previously addressed this question between two nematodes, Caenorhabditis briggsae and C. nigoni, by systematic substitution of various parts of the C. nigoni genome with its C. briggsaes equivalent followed by phenotypic examination. Here we investigate the genetic mechanism of the asymmetric sterility and inviability in the hybrid F1 male and female progeny between the two species. We achieved this through crossing a cohort of C. nigoni strains each carrying a substitution with C. briggsae, which led to differential homozygosity of the C. briggsae substitution in the hybrid progeny. The aggregated substitutions cover 94.6% of the C. briggsae genome, including 100% of the X chromosome. Surprisingly, we identified two C. briggsae X fragments that produced C. nigoni male sterility as a substitution but rescued hybrid F1 sterility in males fathered by C. briggsae, indicating that at least two separate X-autosome interactions are involved in the hybrid male sterility. In addition, we identified multiple genomic intervals on C. briggsae autosomes that can rescue the inviability, but not the sterility, of hybrid F1 males fathered by C. nigoni. Importantly, we isolated a 1.1-Mb genomic interval that specifically interacts with an X-linked introgression, which is essential for hybrid male fertility. We further identified three C. briggsae genomic intervals on the Chromosome I, II and III, respectively that produce inviability in all F1 progeny dependent or independent of the parent-of-origin. The identified interacting loci lays a foundation for their molecular characterization.

evolutionary biology↗

bioRxiv2019

A genome-based species taxonomy of the Lactobacillus Genus Complex

BackgroundThere are over 200 published species within the Lactobacillus Genus Complex (LGC), the majority of which have sequenced type strain genomes available. Although gold standard, genome-based species delimitation cutoffs are accepted by the community, they are seldom checked against currently available genome data. In addition, there are many species-level misclassification issues within the LGC. We constructed a de novo species taxonomy for the LGC based on 2,459 publicly available, decent-quality genomes and using a 94% core nucleotide identity threshold. We reconciled these de novo species with published species and subspecies names by (i) identifying genomes of type strains in our dataset and (ii) performing comparisons based on 16S rRNA sequence identity against type strains. ResultsWe found that genomes within the LGC could be divided into 239 clusters (de novo species) that were discontinuous and exclusive. Comparison of these de novo species to published species lead to the identification of ten sets of published species that can be merged and one species that can be split. Further, we found at least eight genome clusters that constitute new species. Finally, we were able to accurately classify 98 unclassified genomes and reclassify 74 wrongly classified genomes. ConclusionsThe current state of LGC species taxonomy is largely consistent with genome data, but there are some inconsistencies as well as genome misclassifications. These inconsistencies should be resolved to evolve towards a meaningful taxonomy where species have a consistent size in terms of sequence divergence.

bioinformatics↗

bioRxiv2019

Comparative genome analysis of Lactobacillus mudanjiangensis, an understudied member of the Lactobacillus plantarum group

The genus Lactobacillus is known to be extremely diverse and consists of different phylogenetic groups that show a diversity roughly equal to the expected diversity of a typical bacterial genus. One of the most prominent phylogenetic groups within this genus is the Lactobacillus plantarum group which contains the understudied Lactobacillus mudanjiangensis species. Before this study, only one L. mudanjiangensis strain, DSM 28402T, was described but without whole-genome analysis. In this study, three strains classified as L. mudanjiangensis, were isolated from three different carrot juice fermentations and their whole-genome sequence was determined, together with the genome sequence of the type strain. The genomes of all four strains were compared with publicly available L. plantarum group genome sequences. This analysis showed that L. mudanjiangensis harbored the second largest genome size and gene count of the whole L. plantarum group. In addition, all members of this species showed the presence of a gene coding for a putative cellulose-degrading enzyme. Finally, three of the four L. mudanjiangensis strains studied showed the presence of pili on scanning electron microscopy (SEM) images, which were linked to conjugative gene regions, coded on plasmids in at least two of the strains studied. Author summaryLactobacillus mudanjiangensis is an understudied species within the Lactobacillus plantarum group. Since its first description, no other studies have reported its isolation. Here, we present the first four genome sequences of this species, which include the genome sequence of the type strain and three new L. mudanjiangensis strains isolated from fermented carrot juice. The genomes of all four strains were compared with publicly available L. plantarum group genome sequences. We found that this species harbored the second largest genome size and gene count of the whole L. plantarum group. Furthermore, we present the first scanning electron microscopy (SEM) images of L. mudanjiangensis, which showed the formation of pili in three strains that we linked to genes related to conjugation. Finally, we found the presence of a unique putative cellulose-degrading enzyme, opening the door for different industrial applications of these Lactobacillus strains.

microbiology↗

bioRxiv2019

The great tit HapMap project: a continental-scale analysis of genomic variation in a songbird

A major aim of evolutionary biology is to understand why patterns of genomic diversity vary among populations and species. Large-scale genomic studies of widespread species are useful for studying how the environment and demographic history shape patterns of genomic divergence, and with the continually decreasing cost of sequencing and genotyping, such studies are now becoming feasible. Here, we carry out one of the most geographically comprehensive surveys of genomic variation in a wild vertebrate to date; the great tit (Parus major) HapMap project. We screened ca 500,000 SNP markers across 647 individuals from 29 populations, spanning almost the entire geographic range of the European great tit subspecies. We found that genome-wide variation was consistent with a recent colonisation across Europe from a single refugium in South-East Europe, with bottlenecks and reduced genetic diversity in island populations. Differentiation across the genome was highly heterogeneous, with clear "islands of differentiation" even among populations with very low levels of genome-wide differentiation. Low local recombination rate in the genome was a strong predictor of high local genomic differentiation (FST), especially in island and peripheral mainland populations, suggesting that the interplay between genetic drift and recombination is a key driver of highly heterogeneous differentiation landscapes. We also detected genomic outlier regions that were confined to one or more peripheral great tit populations, most likely as a result of recent directional selection at the range edges of this species. Haplotype-based measures of selection were also related to recombination rate, albeit less strongly, and highlighted population-specific sweeps that likely resulted from positive selection. These regions under positive selection contained candidate genes associated with morphology, thermal adaptation and colouration, providing promising avenues for future investigation. Our study highlights how comprehensive screens of genomic variation in wild organisms can provide unique insights into evolution.

evolutionary biology↗