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bioRxiv2023

16S rRNA phylogeny and clustering is not a reliable proxy for genome-based taxonomy in Streptomyces

Although Streptomyces is one of the most extensively studied genera of bacteria, their taxonomy remains contested and is suspected to contain significant species-level misclassification. Resolving the classification of Streptomyces would benefit many areas of study and applied microbiology that rely heavily on having an accurate ground truth classification of similar and dissimilar organisms, including comparative genomics-based searches for novel antimicrobials in the fight against the ongoing antimicrobial resistance (AMR) crisis. To attempt a resolution, we investigate taxonomic conflicts between 16S rRNA and whole genome classifications using all available 48,981 full-length 16S rRNA Streptomyces sequences from the combined SILVA, Greengenes, Ribosomal Database Project (RDP) and NCBI (National Center for Biotechnology Information) databases, and 2,276 publicly available Streptomyces genome assemblies. We construct a 16S gene tree for 14,239 distinct Streptomyces 16S rRNA sequences, identifying three major lineages of Streptomyces, and find that existing taxonomic classifications are inconsistent with the tree topology. We also use these data to delineate 16S and whole genome landscapes for Streptomyces, finding that 16S and whole-genome classifications of Streptomyces strains are frequently in disagreement, and in particular that 16S zero-radius Operational Taxonomic Units (zOTUs) are often inconsistent with Average Nucleotide Identity (ANI)-based taxonomy. Our results strongly imply that 16S rRNA sequence data does not map to taxonomy sufficiently well to delineate Streptomyces species reliably, and we propose that alternative markers should instead be adopted by the community for classification and metabarcoding. As much of current Streptomyces taxonomy has been determined or supported by historical 16S sequence data and may in parts be in error, we also propose that reclassification of the genus by alternative approaches is required. Impact StatementAccurate classification of microbes, usually in the form of taxonomic assignments, provides a fundamental ground truth or reference point for many aspects of applied microbiology including comparative genomics, identification of strains for natural product discovery, and dereplication of strains. Bacteria belonging to the genus Streptomyces are an important source of bioactive metabolites and enzymes in biotechnology, and proper understanding of their phylogeny aids understanding of the evolution of industrially important gene products and metabolites, and prioritization of strains for industrial exploitation. Taxonomic classification in the genus Streptomyces is complex and contested, and there are clear conflicts between taxonomies inferred from 16S rRNA and from whole genome sequences. Despite this, 16S sequence-based classifications are still widely used to infer taxonomic identity, to determine community composition, and to prioritise strains for study. We investigate a diverse and comprehensive set of Streptomyces genomes using whole-genome Average Nucleotide Identity (ANI) and 16S sequence analysis to delineate and compare classifications made using these approaches. We outline the genomic and 16S sequence landscape of Streptomyces, demonstrating that (i) distinct taxonomic species may share identical full-length 16S sequences, and (ii) in some instances, isolates representing the same taxonomic species do not share any common 16S rRNA sequence. Our results strongly imply that 16S rRNA sequence variation does not map to taxonomy sufficiently well to delineate Streptomyces species reliably, and that alternative markers should instead be adopted by the community. Much of current Streptomyces taxonomy has been determined or supported by historical 16S sequence data, and we therefore propose that reclassification within this group by alternative approaches is required. Data summaryAll code, raw and supporting data are publicly available from GitHub (https://github.com/kiepczi/Kiepas_et_al_2023_16S) and Zenodo (https://doi.org/10.5281/zenodo.8223787). The flowchart provided in Supplementary File 28 provides an overview of analysis steps and serves as a guide through Supplementary Files generated during reconstruction of the 16S phylogeny. The flowchart in Supplementary File 29 outlines the workflow processes and supplementary materials used for analysis of 16S rRNA sequences from Streptomyces genomes. Supplementary DataSupplementary File 1: Generate figures using Python and R. ZIP file containing all data, Python and R scripts to generate figures for this manuscript. (ZIP 40.9MB) Supplementary File 2: Raw 16S rRNA public databases. Zip file containing four separate txt files with sequence IDs for public 16S rRNA databases used in this manuscript, and an additional txt file with Greengenes sequence taxonomy information, and a Python script used to map taxonomy information to sequences found in Greengenes v13.5. (ZIP 34.8MB) Supplementary File 3: Filtration of 16S rRNA public databases. Zip file containing Python script used for filtration of the raw databases, and generated outputs. (ZIP 7.2MB) Supplementary File 4: Cleaning of the filtrated 16S rRNA local. Zip file containing all bash and Python scripts used to clean the local full-length 16S rRNA local databases by removing redundant and poor quality 16S rRNA sequences. (ZIP 9MB) Supplementary File 5: Sequence Clustering. Zip file containing a bash script used to cluster the full-length cleaned local 16S rRNA Streptomyces local databases at various thresholds, and provides txt files with accessions for representative sequences, and cluster members for each clustering threshold. (ZIP 40.8MB) Supplementary File 6: Analysis of taxonomic composition for each clustering threshold. Zip file containing Python scripts, NCBI taxonomy input and all outputs generated used to determine the taxonomic composition for each clustering threshold. (ZIP 49.6) Supplementary File 7. Cluster sizes. Empirical cumulative frequency plot showing cluster sizes generated for all clustering thresholds. (PDF 44KB) Supplementary File 8. Cluster taxID abundance. Empirical cumulative frequency plot for unique numbers of taxID present at all clustering thresholds. (PDF 9KB) Supplementary File 9. MSA. Zip file containing all Python and bash scripts, and additional data needed to generate and clean MSA for phylogenetic analysis. (ZIP 4.2MB) Supplementary File 10. Phylogenetic reconstruction. ZIP file containing bash scripts used for phylogenetic reconstruction, and all generated outputs and log files. (ZIP 16.8MB). Supplementary File 11. Collapse branches. ZIP file containing jupyter notebook used for collapsing branches with the same species names, and the collapsed tree in newick format. (ZIP 385KB) Supplementary File 12. Phylogenetic tree. PDF file showing collapsed phylogenetic tree with marked branches with transfer bootstrap expectation support of >= 50%. (PDF 224KB) Supplementary File 13. Phylogenetic tree. PDF file showing collapsed phylogenetic tree showing distribution of Streptomyces albus and Streptomyces griseus. (PDF 229KB) Supplementary File 14. Phylogenetic tree. PDF file showing collapsed phylogenetic tree showing distribution of Streptomyces albulus, Streptomyces lydicus and Streptomyces venezuelae. (PDF 228KB) Supplementary File 15. Phylogenetic tree. PDF file showing collapsed phylogenetic tree showing distribution of Streptomyces clavuligerus and Streptomyces coelicolor. (PDF 227KB) Supplementary File 16. Phylogenetic tree. PDF file showing collapsed phylogenetic tree showing distribution of Streptomyces lavendulae, Streptomyces rimosus and Streptomyces scabiei. (PDF 228KB) Supplementary File 17. Streptomyces genomes. Zip file containing bash scripts used to download Streptomyces genomes, and Python scripts used to check assembly status. The ZIP file also contains two separate txt files with Streptomyces genomes used in this manuscript: one file with all initial candidates, and a second file with replaced genomes. (ZIP 2.6MB) Supplementary File 18. Extraction of full-length and ambiguity free 16S rRNA sequences from Streptomyces genomes. Zip file containing all Python and bash scripts used to extract full-length sequences from the filtered Streptomyces genomes. A single FASTA file with all extracted 16S rRNA sequences, and a single FASTA file with filtered sequences. A txt file with accession of genomes retained in the analysis. (ZIP 742KB) Supplementary File 19. ANI analysis among Streptomyces genomes with identical 16S rRNA sequences. ZIP file containing all Bash and Python scripts used to determine taxonomic boundaries among Streptomyces genomes sharing identical full-length 16S rRNA sequences. All output and pyANI log files. (ZIP 37.1MB) Supplementary File 20. Network analysis of genomes based on shared 16S sequences. ZIP file containing jupyter notebook with NetworkX analysis and all associated output files including. bash script for pyANI analysis runs on each connected component and all associated matrices, heatmaps and log files. (ZIP 29.3MB) Supplementary File 21. Interactive network graph. HTML file containing interactive network graph of genomes sharing common full-length 16S sequences with each node colour corresponding to the number of connections/degrees. (HTML 4.7MB) Supplementary File 22. Interactive network graph. HTML file containing interactive network graph of genomes sharing common full-length 16S sequences showing clique (blue) and non-clique (green) components. (HTML 4.7MB) Supplementary File 23. Interactive network graph. HTML file containing interactive network graph of genomes sharing common full-length 16S sequences showing number of unique genera within each connected component. Each candidate genus is represented as a single node colour within a connected component. (HTML 4.7MB) Supplementary File 24. Interactive network graph. HTML file containing interactive network graph of genomes sharing common full-length 16S rRNA sequences showing number of unique species within each connected component. Each candidate species is represented as a single node colour within a connected component. (HTML 4.7MB) Supplementary File 25 Interactive network graph. HTML file containing interactive network graph of genomes sharing common full-length 16S rRNA sequences showing number of unique NCBI names within each connected component. Each NCBI assigned name is represented as a single node colour within a connected component. Gray nodes represent genomes currently lacking assigned species names. (HTML 4.7MB) Supplementary file 26. Intragenomic 16S rRNA heterogeneity within 1,369 Streptomyces genomes which exclusively contain only full-length and ambiguity symbol-free 16S rRNA sequences. A total of 811 genomes containing single 16S rRNA sequences are not shown. (PDF 8KB) Supplementary File 27. Distribution of 16S copies per genome with a distinction between unique and total copies for genomes at assembly level complete and chromosome. (PDF 7KB) Supplementary File 28. Schematic workflow for construction of the full-length 16S rRNA Streptomyces phylogeny. Each arrow represents a process and is annotated with script used and corresponding supplementary file. Output/data files, and the number of remaining sequences after each step, are indicated by rectangles. The green shading represents a single processing step of collecting and collating 16S database sequences. (PDF 91KB) Supplementary File 29. Schematic representation of the pipeline used to filter publicly available Streptomyces genomes. (PDF 59KB) Supplementary File 30. Sankey plot showing counts of taxonomic names in source databases, assigned at ranks from phylum to genus, to sequences identified with a key word Streptomyces in the taxonomy field. Note that Actinobacteria and Actinobacteriota are synonyms in LPSN for the correct Phylum name Actinomycetota, but that Actinomycetales and Streptomycetales are not taxonomic synonyms for each other. Streptomycetales is synonymous in LPSN with the correct name Kitasatosporales; Actinomycetales is a distinct taxonomic Order. The parent order of the Family Streptomycetaceae in LPSN is Kitasatosporales. (PDF 64KB) Supplementary File 31. Rectangular phylogram of the comprehensive maximum-likelihood tree of the genus Streptomyces based on the 16S sequence diversity of all 5,064 full-length 16S rRNA sequences with 100 TBE values. (PDF 194KB) Supplementary file 32. Genomes sharing identical 16S rRNA sequences are assigned different names in NCBI. A total of 1,030 singleton clusters are not shown. (PDF 8KB) Supplementary File 33. Phylogenetic tree. PDF file showing collapsed phylogenetic tree showing distribution of members of the novel Acintacidiphila genus. (PDF 228KB) Supplementary File 34. Phylogenetic tree. PDF file showing collapsed phylogenetic tree showing distribution of members of the novel Phaeacidiphilus genus. (PDF 228KB) Supplementary File 35. Phylogenetic tree. PDF file showing collapsed phylogenetic tree showing distribution of members of the novel Mangrovactinospora genus. (PDF 228KB) Supplementary File 36. Phylogenetic tree. PDF file showing collapsed phylogenetic tree showing distribution of members of the novel Wenjunlia genus. (PDF 228KB) Supplementary File 37. Phylogenetic tree. PDF file showing collapsed phylogenetic tree showing distribution of members of the novel Streptantibioticus genus. (PDF 228KB)

genomics↗

bioRxiv2019

Comparative genomics analysis reveals high levels of differential DNA transposition among primates

Mobile elements generated via DNA transposition constitute [~]50% of the primate genomes. As a result of past and ongoing activity, DNA transposition is responsible for generating inter- and intra-species genomic variations, and it plays important roles in shaping genome evolution and impacting gene function. While limited analysis of mobile elements has been performed in many primate genomes, a large-scale comparative genomic analysis examining the impact of DNA transposition on primate evolution is still missing.\n\nUsing a bioinformatics comparative genomics approach, we performed analysis of species-specific mobile elements (SS-MEs) in eight primate genomes, which include human, chimpanzee, gorilla, orangutan, green monkey, crab-eating macaque, rhesus monkey, and baboon. These species have good representations for the top two primate families, Hominidae (great apes) and the Cercopithecidae (old world monkeys), for which draft genome sequences are available.\n\nOur analysis identified a total of 230,855 SS-MEs from the eight primate genomes, which collectively contribute to [~]82 Mbp genome sequences, ranging from 14 to 25 Mbp for individual genomes. Several new interesting observations were made based on these SS-MEs. First, the DNA transposition activity level reflected by the numbers of SS-MEs was shown to be drastically different across species with the highest (baboon genome) being more than 30 times higher than the lowest (crab-eating macaque genome). Second, the compositions of SS-MEs, as well as the top active ME subfamilies, also differ significantly across genomes. By the copy numbers of SS-MEs divided into major ME classes, SINE represents the dominant class in all genomes, but more so in the Cercopithecidae genomes than in the Hominidae genomes in general with the orangutan genome being the outliner of this trend by having LINE as the dominant class. While AluY represents the major SINE groups in the Hominidae genomes, AluYRa1 is the dominant SINE in the Cercopithecidae genomes. For LINEs, each Hominidae genome seems to have a unique most active L1 subfamily, but all Cercopithecidae genomes have L1RS2 as the most active LINEs. While genomes with a high number of SS-MEs all have one or more very active ME subfamilies, the crab-eating macaque genome, being the one with an extremely low level of DNA transposition, has no single ME class being very active, suggesting the existence of a genome-wide mechanism suppressing DNA transposition. Third, DNA transposons, despite being considered dead in primate genomes, were in fact shown to have a certain level of activity in all genomes examined with a total of [~]2,400 entries as SS-MEs. Among these SS-MEs, at least 23% locate to genic regions, including exons and regulatory elements, presenting significant potentials for their impact on gene function. Very interestingly, our data demonstrate that, among the eight primates included in this study, the human genome is shown to be the most actively evolving genome via DNA transposition as having the highest most recent activity of many ME subfamilies, notably the AluYa5/Yb8/Yb9, L1HS, and SVA-D subfamilies.\n\nRepresenting the first of its kind, our large-scale comparative genomics study has shown that mobile elements evolved quite differently among different groups and species of primates, indicating that differential DNA transposition has served as an important mechanism in primate evolution.

evolutionary biology↗

bioRxiv2016

Animals transcribe at least half of the genome

One central goal of genome biology is to understand how the usage of the genome differs between organisms. Our knowledge of genome composition, needed for downstream inferences, is critically dependent on gene annotations, yet problems associated with gene annotation and assembly errors are usually ignored in comparative genomics. Here we analyze the genomes of 68 species across all animal groups and some single-cell eukaryotes for general trends in genome usage and composition, taking into account problems of gene annotation. We show that, regardless of genome size, essentially all animals have comparable amounts of introns and intergenic sequence, with nearly all deviations dominated by increased intergenic sequence. Genomes of model organisms have ratios much closer to 1:1, suggesting that the majority of published genomes of non-model organisms are underannotated and consequently omit substantial numbers of genes, with likely negative impact on evolutionary interpretations. Finally, our results also indicate that most animals transcribe half or more of their genomes arguing against differences in genome usage between animal groups, and also suggesting that the transcribed portion is more dependent on genome size than previously thought.\n\nAuthors SummaryWithin our anthropocentric genomic framework, many analyses tends to try to define humans, mammals, or vertebrates relative to the so-called \"lower\" animals. This implicitly posits that vertebrates are complex organisms with large genomes and invertebrates are simple organisms with small genomes. This has the problem that genome size is therefore presumed to correlate with complexity and ignores any unknown complexity of vast numbers of invertebrate groups, many with large genomes. Animals vary widely in genome size, by almost three orders of magnitude, but when sequencing new animal genomes preference is given to those with smaller genomes for reasons of cost. In trying to understand how genomes are used in general, there is an added layer of complication from quality of the assembly and annotation. We have examined genome usage across a wide range of animals and have described ways to account for errors of low-quality annotations. We also show that the genomes of invertebrates and vertebrates are not so different, and that when large-genome invertebrates are added, genome size alone appears to be defining the fraction of the genome that is genes.

Genomics↗

bioRxiv2023

Genomic characterization of the C. tuberculostearicum species complex, a ubiquitous member of the human skin microbiome

Corynebacterium is a predominant genus in the skin microbiome, yet its genetic diversity on skin is incompletely characterized and lacks a comprehensive set of reference genomes. Our work aims to investigate the distribution of Corynebacterium species on the skin, as well as to expand the existing genome reference catalog to enable more complete characterization of skin metagenomes. We used V1-V3 16S rRNA gene sequencing data from 14 body sites of 23 healthy volunteers to characterize Corynebacterium diversity and distribution across healthy human skin. Corynebacterium tuberculostearicum is the predominant species found on human skin and we identified two distinct C. tuberculostearicum ribotypes (A & B) that can be distinguished by variation in the 16S rRNA V1-V3 sequence. One is distributed across all body sites and the other found primarily on the feet. We performed whole genome sequencing of 40 C. tuberculostearicum isolates cultured from the skin of five healthy individuals across seven skin sites. We generated five closed genomes of diverse C. tuberculostearicum which revealed that C. tuberculostearicum isolates are largely syntenic and carry a diversity of methylation patterns, plasmids and CRISPR/Cas systems. The pangenome of C. tuberculostearicum is open with a core genome size of 1806 genes and a pangenome size of 5451 total genes. This expanded pangenome enabled the mapping of 24% more C. tuberculostearicum reads from shotgun metagenomic datasets derived from skin body sites. Finally, while the genomes from this study all fall within a C. tuberculostearicum species complex, the ribotype B isolates may constitute a new species. IMPORTANCEAmplicon sequencing data combined with isolate whole genome sequencing has expanded our understanding of Corynebacterium on the skin. Human skin is characterized by a diverse collection of Corynebacterium species but C. tuberculostearicum predominates many sites. Our work supports the emerging idea that C. tuberculostearicum is a species complex encompassing several distinct species. We produced a collection of genomes that help define this complex including a potentially new species which we are calling C. hallux based on a preference for sites on the feet, whole-genome average nucleotide identity, pangenomics and growth in skin-like media. This isolate collection and high-quality genome resource sets the stage for developing engineered strains for both basic and translational clinical studies. Microbiomes are shaped by taxa that are both characteristic to those sites and functionally important to that community. The genus Corynebacterium is one such taxa for the human skin and nares. Foundational studies using 16S rRNA gene sequencing and shotgun metagenomics by our lab (1, 2) and others (3) have established Corynebacterium as common members of the skin microbiome. While Corynebacterium have been positively correlated with the resolution of dysbiosis associated with eczema flares (4), the importance of the Corynebacterium spp. is less defined for skin disease severity in primary immune deficient patients (5, 6). Corynebacterium spp. are predominant members of the human aerodigestive tract microbiome (nares, oral cavity and respiratory tract) (3) and participate in microbe-microbe interactions with members of nasal microbiome (7, 8). Corynebacterium have been shown to engage with the host immune system, specifically C. accolens-promoted IL23-dependent inflammation in mice on a high-fat diet (9). C. bovis and C. mastiditis have been shown to predominate the microbiome of a ADAM10-deficient mouse model (10) as well as an ADAM17-deficient mouse model of eczema (11). Finally, C. tuberculostearicum has been shown to induce inflammation in human epidermal keratinocyte cell cultures (12). These studies establish Corynebacterium spp. as key members of the skin microbiome capable of both microbe-microbe and microbe-host interactions. A critical resource for understanding the biology of Corynebacterium on the skin is a robust collection of complete reference genomes, including isolates collected from a variety of individuals and body sites. Previously published genome collections from skin- or nares-resident species include Staphylococcus epidermidis (13), Cutibacterium acnes (14) and the recent comparative analysis of Dolosigranulum pigrum (15). Of note, while emerging bioinformatic methods and pipelines are now being employed to extract nearly-complete genomes (MAGs) from metagenomic assemblies of skin samples (16), MAGs are not yet a substitute for genomes from cultured isolates to understand strain level or pangenomic diversity. In addition to functional prediction, comparative genomics is increasingly being used to augment conventional microbiological methods to define or redefine taxonomic boundaries (17, 18), as well as describe the full extent of diversity within these boundaries (19). A pangenome, which encompasses the complete set of genes present within a set of genome sequences, enables the characterization of gene-level heterogeneity within a taxonomic group. The pangenome is commonly subdivided into the core genome, referring to genes present in all strains, and the accessory or dispensable genome, referring to those present in only one or some isolates. (The accessory pangenome can be further subdivided to reflect a wider range of gene uniqueness, e.g. singletons.) Thorough characterization of taxa is limited by the availability of representative and high-quality genome assemblies. Unfortunately, with the exceptions of clinically relevant Corynebacterium spp. (e.g., C. diphtheriae, C. striatum and C. pseudotuberculosis), the genus is inadequately sequenced, with 75% of species having fewer than six genomes. This includes common skin-associated species like C. tuberculostearicum with just five unique isolate genomes, only two of which are from skin. This work seeks first to characterize the distribution of Corynebacterium across 14 skin sites from 23 healthy volunteers. The second goal of this work focuses on what we identify as the predominant skin Corynebacterium species, C. tuberculostearicum. We have sequenced 23 distinct C. tuberculostearicum strains (n=40 genomes before dereplication), a five-fold increase in the number of publicly available, unique genomes (n=5). In addition to short-read assemblies, we generated five complete genomes which, along with the type strain (DSM44922), demonstrate that C. tuberculostearicum genomes are largely syntenic and carry a number of methylation systems as well as a CRISPR/Cas system. Genes from the C. tuberculostearicum genomes in our collection fall into 5451 gene clusters comprising the species pangenome. This expanded pangenome, as compared to existing public references, improved the mapping of C. tuberculostearicum metagenomic reads from unrelated healthy volunteers. In addition, we have identified a distinct C. tuberculostearicum clade that is highly enriched on the feet that may represent a new species, tentatively designated Corynebacterium hallux.

microbiology↗

bioRxiv2026

Defining Aeromonas species boundaries with large-scale average nucleotide identity analyses and genomic phylogeny

Aeromonas taxonomy has long been complicated by overlapping phenotypic, biochemical, and protein profiles. Here, we establish a robust genome-based framework for Aeromonas genomic species delineation. We analysed average nucleotide identity (ANI) across 4,366 available Aeromonas genomes and demonstrated that at 96% ANI threshold, skANI and fastANI generated too many clusters (65 and 57 respectively) and these clusters were not supported by core genome phylogeny. We identified 95.4% skANI (equivalent to 95.6% fastANI) as an operational threshold for the delineating Aeromonas genomic species. Using the 95.4% skANI threshold, we identified 44 ANI clusters among the 4,366 genomes, of which 43 clusters were genomic species supported by the core-genome phylogeny. Thirty-four of the 43 genomic species corresponded to existing taxonomic species, whilst the remaining nine are currently not recognised as taxonomic species. All recognised taxonomic species represented in the dataset retained their existing species designation except Aeromonas mytilicola, which was not separated from Aeromonas rivipollensis in both ANI clusters and the core-genome phylogeny. The digital DNA-DNA hybridisation (dDDH) values between the genomic species were below 70%, further supporting genomic species delineation. We further developed AeromonasGStyper, a genomic species typing tool that assigns query genomes based on ANI similarity to medoid genomes. In conclusion, this study establishes a genomic species framework for genome-based classification of Aeromonas and provides a practical approach for future genomic surveillance. Impact StatementAeromonas species have gained increased attention as emerging human enteric pathogens. Aeromonas taxonomy has long been complicated by overlapping phenotypic, biochemical, and protein profiles. Although a 96% average nucleotide identity (ANI) threshold was proposed previously for Aeromonas species delineation, analysis of 4,366 Aeromonas genomes demonstrated that this threshold generated excessive genomic clusters that were not supported by the core-genome phylogeny. We identified 95.4% skANI (equivalent to 95.6% fastANI) as an operational threshold for delineating Aeromonas genomic species, supported by core-genome phylogeny and digital DNA-DNA hybridisation (dDDH). The framework identified 43 genomic species, including 34 corresponding to recognised taxonomic species and nine genomic species that do not correspond to currently recognised taxonomic species. In addition, our data showed that Aeromonas mytilicola was not separated from Aeromonas rivipollensis by ANI clustering and core-genome phylogeny, supporting further taxonomic reassessment of the distinction between these two species.

microbiology↗

bioRxiv2022

Human Papillomavirus Genome Copy Number Is Maintained by S Phase Amplification, Leakage to the Cytosol during Mitosis and Lysosomal Degradation in G1 Phase

The current model for human papillomavirus (HPV) replication is comprised of three modes of replication. Following infectious delivery, the viral genome is amplified during the establishment phase to reach up to some hundred copies per cell. HPV genome copy number remains constant during the maintenance stage. Differentiation of infected cells induces HPV genome amplification. Using highly sensitive in situ hybridization (DNAscope) and freshly HPV16-infected as well as established HPV16-positive cell lines, we observed that viral genome is amplified in each S phase of undifferentiated keratinocytes cultured as monolayers. Nuclear viral genome copy number is reset to pre-S phase levels during mitosis. The majority of viral genome fails to tether to host chromosomes and is lost to the cytosol. Cytosolic viral genome gradually decreases during cell cycle progression. Loss of cytosolic genome is blocked in presence of NH4Cl or other drugs interfering with lysosomal acidification, suggesting the involvement of autophagy in viral genome degradation. These observations were also made with HPV31 cell lines obtained from patient samples. Cytosolic viral genome was not detected in UMSCC47 cells carrying integrated HPV16 DNA. Analyses of organotypic raft cultures derived from keratinocytes harboring episomal HPV16 revealed the presence of cytosolic viral genome as well. We conclude that HPV maintains viral genome copy number by balancing viral genome amplification during S phase with loss of viral genome that is lost to the cytosol during mitosis. It seems plausible that restrictions to viral genome tethering to mitotic chromosomes resets genome copy number in each cell cycle. IMPORTANCEHPV genome maintenance is currently being thought to be achieved by regulating expression and activity of viral replication factors E1 and E2. In addition, the E8^E2 repressor has been shown to be important for restricting genome copy number by competing with E1 and E2 for binding to the viral origin of replication and by recruitment of repressor complexes. Herein, we demonstrate that HPV viral genome is amplified in each S phase. Nuclear genome copy number is reset during mitosis by a failure of the majority of genomes to tether to mitotic chromosomes. Rather, they accumulate in the cytoplasm of freshly divided cells. Cytosolic viral DNA is quickly degraded in G1 in a lysosome dependent manner contributing to the genome copy reset. Our data imply that the mode of replication during establishment and maintenance is the same and further suggest that restrictions to genome tethering significantly contributes to viral genome maintenance.

microbiology↗

bioRxiv2023

skDER: microbial genome dereplication approaches for comparative and metagenomic applications

An abundance of microbial genomes have been sequenced in the past two decades. For fundamental comparative genomic investigations, where the goal is to determine the major gain and loss events shaping the pangenome of a species, it is often unnecessary and computationally onerous to include all available genomes in studies. In addition, over-representation of specific lineages due to sampling and sequencing bias can have undesired effects on evolutionary analyses. To assist users with genomic dereplication, selecting a subset of representative genomes, for downstream comparative genomic investigations, we developed skDER & CiDDER (https://github.com/raufs/skDER). skDER combines recent advances to efficiently estimate average nucleotide identity (ANI) between thousands of microbial genomes with two efficient algorithms for genomic dereplication. Further, CiDDER implements an approach whereby protein clusters are determined across all genomes and genomes are iteratively selected as representatives until a user-defined saturation of the total protein space is achieved. To support ease of use, several auxiliary functionalities are implemented within the two programs, including arguments to: (i) test the number of representative genomes resulting from a variety of clustering parameters, (ii) automate downloading of genomes belonging to a bacterial species or genus by name, (iii) cluster non-representative genomes to their closest representative genomes, and (iv) automatically filter predicted plasmids and phages prior to dereplication. We further assess the effects of filtering mobile genetic elements (MGEs) on ANI and alignment fraction (AF) estimates between pairs of genomes and find that MGEs tend to slightly deflate both metrics in one species. DATA SUMMARYskDER and CiDDER are provided as open-source software implemented in Python and C++ on Github: https://github.com/raufs/skDER; with version updates tracked on Zenodo: https://zenodo.org/records/138877101. Installation of the software is supported via both Bioconda2 and Docker. Additional code and data for analyses presented in this manuscript can be found on Zenodo at: https://zenodo.org/records/138918003. Pre-computed representative genomes selected by skDER (v1.0.7) for 18 common bacterial taxonomic groups, referencing classifications from GTDB release 2144, are also provided on Zenodo at: https://zenodo.org/records/100412035. IMPACT STATEMENTDue to the increased availability of genomes for certain microbial species, performing fundamental comparative genomic investigations has become restricted to those with access to more advanced computational infrastructure. Genomic dereplication, the process of selecting distinct representative genomes to capture the breadth of a taxonomic group, thus presents a valuable solution to overcome this embarrassment of riches. Specifically, genomic dereplication allows simplifying the scale of comparative investigations while minimizing the risk of biasing analyses to specific lineages, which might be overrepresented in genomic databases. We present here two programs for genomic dereplication, one based on ANI-inference, skDER, and the other based on assessing the saturation of total coding-genes sampled, CIDDER. These tools are implemented with a variety of auxiliary options and designed for ease of use.

bioinformatics↗

bioRxiv2021

Role of spatial patterning of N-protein interactions in SARS-CoV-2 genome packaging

Viruses must efficiently and specifically package their genomes while excluding cellular nucleic acids and viral sub-genomic fragments. Some viruses use specific packaging signals, which are conserved sequence/structure motifs present only in the full-length genome. Recent work has shown that viral proteins important for packaging can undergo liquid-liquid phase separation (LLPS), where one or two viral nucleic acid binding proteins condense with the genome. The compositional simplicity of viral components lends itself well to theoretical modeling compared to more complex cellular organelles. Viral LLPS can be limited to one or two viral proteins and a single genome that is enriched in LLPS-promoting features. In our previous study, we observed that LLPS-promoting sequences of SARS-CoV-2 are located at the 5' and 3' ends of the genome, whereas the middle of the genome is predicted to consist mostly of solubilizing elements. Is this arrangement sufficient to drive single genome packaging, genome compaction, and genome cyclization? We addressed these questions using a coarse-grained polymer model, LASSI, to study the LLPS of nucleocapsid protein with RNA sequences that either promote LLPS or solubilization. With respect to genome cyclization, we find the most optimal arrangement restricts LLPS-promoting elements to the 5' and 3' ends of the genome, consistent with the native spatial patterning. Genome compaction is enhanced by clustered LLPS-promoting binding sites, while single genome packaging is most efficient when binding sites are distributed throughout the genome. These results suggest that many and variably positioned LLPS-promoting signals can support packaging in the absence of a singular packaging signal which argues against necessity of such a feature. We hypothesize that this model should be generalizable to multiple viruses as well as cellular organelles like paraspeckles, which enrich specific, long RNA sequences in a defined arrangement. Statement of significanceThe COVID-19 pandemic has motivated research of the basic mechanisms of coronavirus replication. A major challenge faced by viruses such as SARS-CoV-2 is the selective packaging of a large genome in a relatively small capsid while excluding host and sub-genomic nucleic acids. Genomic RNA of SARS-CoV-2 can condense with the Nucleocapsid (N-protein), a structural protein component critical for packaging of many viruses. Notably, certain regions of the genomic RNA drive condensation of N-protein while other regions solubilize it. Here, we explore how the spatial patterning of these opposing elements promotes single genome compaction, packaging, and cyclization. This model informs future in silico experiments addressing spatial patterning of genomic features that are experimentally intractable because of the length of the genome.

biophysics↗

bioRxiv2025

amr.watch - monitoring antimicrobial resistance trends from global genomics data

BackgroundWhole genome sequencing (WGS) is increasingly supporting routine pathogen surveillance at local and national levels, providing comparable data that can inform on the emergence and spread of antimicrobial resistance (AMR) globally. However, the potential for shared WGS data to guide interventions around AMR remains under-exploited, in part due to challenges in collating and transforming the growing volumes of data into timely insights. We present an interactive platform, amr.watch (https://amr.watch), that enables interrogation of AMR trends from public WGS data on an ongoing basis to support research and policy. MethodsThe amr.watch platform incorporates, analyses and visualises high-quality WGS data from WHO-defined priority bacterial pathogens. Analytics are performed using community-standard methods with bespoke species-specific curation of AMR mechanisms. FindingsBy 31 March 2025, amr.watch included data from 620,700 pathogen genomes with geotemporal information, with highly variable representation of different species and geographic regions. By integrating WGS data with sampling information, amr.watch enables users to assess geotemporal trends among genotypic variants (e.g. sequence types) and AMR mechanisms, with implications for interventions including antimicrobial prescribing and drug and vaccine development. Interpretationamr.watch is an information platform for scientists and policy-makers delivering ongoing situational awareness of AMR trends from genomic data. As broad adoption of WGS continues, amr.watch is positioned to monitor both pathogen populations and our global efforts in genomic surveillance, guiding control strategies tailored to each pathogens characteristics. FundingUK National Institute for Health Research & Gates Foundation. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSWhole genome sequencing (WGS) approaches enable us to track the spread of bacterial pathogens and antimicrobial resistance (AMR) with high resolution at local, national and global levels. To date, genomic studies assessing AMR dynamics have largely used retrospective data collected for specific research agendas. However, the growing volumes of publicly-shared WGS data, generated increasingly from routine surveillance, provide improved power to detect novel trends and guide interventions. Efforts to collate public bacterial genome data exist, such as AllTheBacteria, although these are aimed at the research community and do not facilitate data usage and interpretation by non-genomics experts, particularly those in public health. To our knowledge, no platforms exist for readily interrogating AMR dynamics from continuously updated public genome data across the array of different WHO priority bacterial pathogens. Of note, however, TyphiNet provides an interactive online dashboard for examining AMR trends exclusively from Salmonella Typhi using a periodically-updated data set from the Pathogenwatch platform. These findings are based on searching PubMed without language restrictions from Jan 1 2000 to December 31 2024, using terms related to "genomic surveillance" and "antimicrobial resistance". Added value of this studyWe have developed amr.watch which, to our knowledge, is the first platform that enables ongoing analysis and visualisation of AMR trends from public genome data across the spectrum of WHO priority bacterial pathogens via an accessible interface. Crucially, the platform incorporates processed genome data via a live always-on stream, enabling insights that are delayed only by the time to data deposition. We reviewed public genomes with available geotemporal information up until 31 March 2025, providing a contemporary global landscape of pathogen genome sequencing. While the number of genomes available annually grew over five-fold globally between 2010 and 2018, we also revealed the extent of differences in geographic representation, with 89.6% of genomes originating from high-income countries and 89 countries contributing no genome data from the priority pathogens. Implications of all the available evidenceRapid generation and sharing of global genome data enables us to more precisely track the spread and define the characteristics of contemporary circulating resistant pathogens. The amr.watch platform provides a solution for retrieving, curating and translating shared genomic data into relevant insights that are accessible and actionable by diverse stakeholders. It thereby forms a basis for monitoring progress in surveillance efforts, alerting on ongoing population changes and guiding enhanced precision for surveillance and interventional development. Additionally, our review of available bacterial genome data highlights the need for additional efforts to increase and sustain the implementation of genomic surveillance of AMR globally, and improve the timely sharing of WGS data and its associated metadata.

genomics↗

bioRxiv2020

Aquatic insects are dramatically underrepresented in genomic research

Aquatic insects comprise 10% of all insect diversity, can be found on every continent except Antarctica, and are key components of freshwater ecosystems. Yet aquatic insect genome biology lags dramatically behind that of terrestrial insects. If genomic effort was spread evenly, one aquatic insect genome would be sequenced for every [~]9 terrestrial insect genomes. Instead, [~]24 terrestrial insect genomes have been sequenced for every aquatic insect genome. This discrepancy is even more dramatic if the quality of genomic resources is considered; for instance, while no aquatic insect genome has been assembled to the chromosome level, 29 terrestrial insect genomes spanning four orders have. We argue that a lack of aquatic insect genomes is not due to any underlying difficulty (e.g., small body sizes or unusually large genomes) yet it is severely hampering aquatic insect research at both fundamental and applied scales. By expanding the availability of aquatic insect genomes, we will gain key insight into insect diversification and empower future research for a globally important taxonomic group. Simple SummaryAquatic insects comprise 10% of all insect diversity, can be found on every continent except Antarctica, and are key components of freshwater ecosystems. Yet aquatic insect genome biology lags dramatically behind that of terrestrial insects. If genomic effort was spread evenly, one aquatic insect genome would be sequenced for every [~]9 terrestrial insect genomes. Instead, [~]24 terrestrial insect genomes have been sequenced for every aquatic insect genome. We argue that the limited availability of aquatic insect genomes is not due to practical limitations--e.g., small body sizes or overly complex genomes--but instead reflects a lack of research interest. We call for targeted efforts to expand the availability of aquatic insect genomic resources to gain key molecular insight into insect diversification and empower future research.

genomics↗

bioRxiv2019

Transposable elements contribute to dynamic genome content in maize

Transposable elements (TEs) are ubiquitous components of eukaryotic genomes and can create variation in genomic organization. The majority of maize genomes are composed of TEs. We developed an approach to define shared and variable TE insertions across genome assemblies and applied this method to four maize genomes (B73, W22, Mo17, and PH207). Among these genomes we identified 1.6 Gb of variable TE sequence representing a combination of recent TE movement and deletion of previously existing TEs. Although recent TE movement only accounted for a portion of the TE variability, we identified 4,737 TEs unique to one genome with defined insertion sites in all other genomes. Variable TEs are found for all superfamilies and are distributed across the genome, including in regions of recent shared ancestry among individuals. There are 2,380 genes annotated in the B73 genome located within variable TEs, providing evidence for the role of TEs in contributing to the substantial differences in gene content among these genotypes. The large scope of TE variation present in this limited sample of temperate maize genomes highlights the major contribution of TEs in driving variation in genome organization and gene content. Significance StatementThe majority of the maize genome is comprised of transposable elements (TEs) that have the potential to create genomic variation within species. We developed a method to identify shared and non-shared TEs using whole genome assemblies of four maize inbred lines. Variable TEs are found throughout the maize genome and in comparisons of any two genomes we find ~20% of the genome is due to non-shared TEs. Several thousand maize genes are found within TEs that are variable across lines, highlighting the contribution of TEs to gene content variation. This study creates a comprehensive resource for genomic studies of TE variability among four maize genomes, which will enable studies on the consequences of variable TEs on genome function.

genomics↗

bioRxiv2024

Comparative genomics reveals intra and inter species variation in the pathogenic fungus Batrachochytrium dendrobatidis

The Global Panzootic Lineage (GPL) of the amphibian pathogen Batrachochytrium dendrobatidis (Bd) has been described as a main driver of amphibian extinctions on nearly every continent. Near complete genomes of three Bd-GPL strains have enabled studies of the pathogen but the genomic features that set Bd-GPL apart from other B. dendrobatidis lineages is not well understood due to a lack of high-quality genome assemblies and annotations from other lineages. We used Oxford Nanopore Technologies (ONT) DNA sequencing to assemble high-quality genomes of three Bd-BRAZIL isolates and one non-pathogen outgroup species Polyrhizophydium stewartii (Ps) strain JEL0888 and compared these to genomes of previously sequenced Bd-GPL strains. The Bd-BRAZIL assemblies range in size between 22.0 and 26.1 Mb and encode 8495-8620 protein-coding genes for each strain. A pangenome is defined as all the genes in a species including the core genome, genes found in every strain, and the accessory genome, genes found in only some strains (Brockhurst et al. 2019). To date, a comprehensive analysis identifying the core and accessory genes within B. dendrobatidis has not been conducted. Furthermore, while previous studies have examined the gene transcription profiles of Bd-GPL and Bd-BRAZIL strains (McDonald et al. 2020), they do not account for the genomic differences between these strains. Our pangenome analysis provides insight into shared and lineage-specific gene content and how B. dendrobatidis genotype affects recovery of RNAseq transcripts from different strains. We hypothesize that gene content differences exist between the B. dendrobatidis lineages and genomic differences, such as gene family expansions or gene sequence variation, affect alignment and enumeration of transcriptomic data when relying on a single reference genome. The pangenome analysis revealed a core genome consisting of 6278 conserved gene families, and an accessory genome with 202 Bd-BRAZIL and 172 Bd-GPL specific gene families. We discovered gene copy number differences in five pathogenicity gene families: M36 Peptidase, Crinkler Necrosis Genes (CRN), Aspartyl Peptidase, Carbohydrate-Binding Module-18 (CBM18), and S41 Protease, between Bd-BRAZIL and Bd-GPL strains. However, none of the five families were expanded in Bd-GPL compared to Bd-BRAZIL strains. Comparison between the Batrachochytrium genus and two closely related non-pathogenic saprophytic chytrids identified differences in sequence and protein domain counts. We further test these new Bd-BRAZIL genomes to assess their utility as reference genomes for transcriptome alignment and analysis. Our analysis examines the genomic variation between strains in Bd-BRAZIL and Bd-GPL and offers insights into the application of these genomes as reference genomes for future studies. SignificanceThe geographically defined enzootic populations of amphibian pathogen Batrachochytrium dendrobatidis harbor gene context variation revealed in pan-genome analyses. Long read sequencing is required to fully capture this diversity as some recently duplicated and potential virulence gene families are undercounted in short-read only genome assemblies. This genetic variation can impact estimates of gene expression differences between strains if a single genome reference is used. It is necessary to consider the pan-genome diversity of the multiple lineages of this important amphibian pathogen and perhaps other fungal pathogens when engaging in studies of adaptation, virulence, and comparative biology of a species.

microbiology↗

bioRxiv2024

Low-cost and highly efficient generation of near-complete bacterial pathogen genomes by TELL-Seq

Recent evidences suggest that de novo genome assembly can provide additional insights into genomic variation landscape beyond short-read NGS analyses. Despite the advent of longread sequencing technologies, generating high-quality bacterial genome assemblies remains expensive and requires high-quality and large quantities of DNA. TELL-Seq, an emerging linked-read technology, has been reported as a low-cost alternative for producing nearcomplete genome assembly in certain bacterial species. However, a systematic assessment of its performance and characteristics in de novo bacterial genome assembly has not yet been conducted. To address this, we benchmarked TELL-Seq using a set of clinical and standard bacterial pathogens with a wide range of genome size (2.0 6.4 Mbp), GC-content (32% - 68%), genome complexity (mappability from 0.15 to 0.60) and Gram types. Our findings indicate that, with 1/6 cost and 1/15,000 of DNA compared to PacBio HiFi, TELL-Seq could generate as high quality near-complete bacterial genome assemblies. The optimal depth for assembly is around 200x, as increased depth improves contiguity and completeness, though quality plateaus at 250x. In general, genome complexity was significantly correlated with assembly quality, but GC-content was not. Nevertheless, genomes with extreme GC-content may require higher depths for accurate assembly. Our study suggests that TELL-Seq could be a scalable method for large-scale bacterial genomic surveys. The data generated from this study could serve as a benchmark dataset for further algorithm development. Impact StatementAn increasing body of literature have shown that de novo genome assembles could provide additional insights into genomic variation beyond standard short-read technologies. Its particularly useful in clinical and epidemiological studies of bacteria, due to high variable nature of their genomes. Although great advances have been made in long-read sequencing technology, its still expensive with high requirement over sample DNAs quality and quantity, limiting its adoption in large-scale studies or surveys. Linked-read technology is an inexpensive alternative which used single molecule barcoding to mark origin of reads for better scaffolding of contigs assembled from normal short reads. TELL-Seq, as one of these promising technologies, was reported to be able to inexpensively and efficiently generate near-complete bacterial de novo genomic assemblies with low DNA inputs. Theres not yet a systematic benchmarking of this technology in bacterial pathogens. In this study, a set of clinically important bacterial pathogens with a wide spectrum of GC-content, genome size, genome complexity, and Gram Type were used to investigate the performance of TELL-Seq technology in de novo genome assembly. We found that, with about 1/6 of cost and 1/15000 of DNA quantity, it could generate comparable de novo assemblies compared to standard reference genomes or PacBio HiFi assemblies, while beating conventional short-read assemblies. We found several key factors, such as genome complexity, and sequencing depth, which would impact the quality of the assembly. Our study suggests that a sequencing depth of 200x is sufficient to achieve satisfactory results. Our study has revealed characteristics of this technology in bacterial genome assembly and paved the way for its large-scale application in clinical or epidemiological surveys. Data SummaryO_LIRaw sequencing data for all isolates have been deposited to SRA, accessible through these NCBI BioProject PRJNA1133244. C_LIO_LIA full list of SRA BioSample accession numbers are available in Tab S5. C_LIO_LIAssemblies and analyses companion this paper is available at: https://github.com/x-lab/tellseq_bacteria C_LI

bioinformatics↗

bioRxiv2021

Large-scale comparative genomics of Salmonella enterica to refine the organization of the global Salmonella population structure

1.Since the introduction of the White-Kauffmann-Le Minor (WKL) scheme for Salmonella serotyping, the nomenclature remains the most widely used for reporting the disease prevalence of Salmonella enterica across the globe. With the advent of whole genome sequencing (WGS), traditional serotyping has been increasingly replaced by in-silico methods that couple the detection of genetic variations in antigenic determinants with sequence-based typing. However, despite the integration of genomic-based typing by in-silico serotyping tools such as SeqSero2 and SISTR, in-silico serotyping in certain contexts remains ambiguous and insufficiently informative due to polyphyletic serovars. Furthermore, in spite of the widespread acknowledgement of polyphyly from genomic studies, the serotyping nomenclature remains unaltered. To prompt refinements to the Salmonella typing nomenclature for disease reporting, we herein performed a systematic characterization of putative polyphyletic serovars and the global Salmonella population structure by comparing 180,098 Salmonella genomes (representing 723 predicted serovars) from GenomeTrakr and PubMLST databases. We identified a range of core genome MLST typing thresholds that result in stable population structure, potentially suitable as the foundation of a genomic-based typing nomenclature for longitudinal surveillance. From the genomic comparisons of hundreds of predicted serovars, we demonstrated that in-silico serotyping classifications do not consistently reflect the population divergence observed at the genomic level. The organization of Salmonella subpopulations based on antigenic determinants can be confounded by homologous recombination and niche adaptation, resulting in shared classification of highly divergent genomes and misleading distinction between highly similar genomes. In consideration of the pivotal role of Salmonella serotyping, a compendium of putative polyphyletic serovars was compiled and made publicly available to provide additional context for future interpretations of in-silico serotyping results in disease surveillance settings. To refine the typing nomenclatures used in Salmonella surveillance reports, we foresee an improved typing scheme to be a hybrid that integrates both genomic and antigenic information such that the resolution from WGS is leveraged to improve the precision of subpopulation classifications while preserving the common names defined by the WKL scheme. Lastly, we stress the importance of controlled vocabulary integration for typing information in open data settings in order for the global Salmonella population dynamics to be fully trackable. 2. Impact StatementSalmonella enterica (S. enterica) is a major foodborne pathogen responsible for an annual incidence rate of more than 90 million cases of foodborne illnesses worldwide. To surveil the high order Salmonella lineages, compare disease prevalence across jurisdictions worldwide, and inform risk assessments, in-silico serotyping has been established as the gold standard for typing the bacteria. However, despite previous Salmonella genomic studies reporting discordance between phylogenomic clades and serovars, refinements have yet been made to the serotyping scheme. Here, we analyzed over 180,000 Salmonella genomes representing 723 predicted serovars to subdivide the population into evolutionarily stable clusters in order to propose a stable organization of the Salmonella population structure that can form the basis of a genomic-based typing scheme for the pathogen. We described numerous instances in which genomes between serotypes are more similar than genomes within a serotype to reflect the inconsistencies of subpopulation classifications based on antigenic determinants. Moreover, we found inconsistencies between predicted serovars and reported serovars which highlighted potential errors in existing in-silico serotyping tools and the need to implement controlled vocabularies for reporting Salmonella subtypes in public databases. The findings of our study aim to motivate the future development of a standardized genomic-based typing nomenclature that more accurately captures the natural populations of S. enterica. 3. Data SummaryThe assembly accession numbers of the genomes analyzed in this study (n = 204,952) and the associated metadata (e.g. sampling location, collection date, FTP address for retrieval) are documented in Table S1. The GenomeTrakr genomes were retrieved from the National Center for Biological Information GenBank database. The PubMLST genomes were retrieved using the BIGSdb API.

genomics↗

bioRxiv2022

A chromosome level genome assembly of longnose gar, Lepisosteus osseus

Holosteans (gars and bowfins) represent the sister lineage to teleost fishes, the latter being a clade that comprises over half of all living vertebrates and includes important models for comparative genomics and human health. A major distinction between the evolutionary history of teleosts and holosteans is that all teleosts experienced a genome duplication event in their early evolutionary history. As holostean genomes did not undergo a round of genome duplication, they have been heralded as a means to bridge teleost models to other vertebrate genomes. However, only three species of holosteans have been genome sequenced to date and sequencing of more species is needed to fill sequence sampling gaps and provide a broader comparative basis for understanding holostean genome evolution. Here we report the first high quality reference genome assembly and annotation of the longnose gar (Lepisosteus osseus). Our final assembly consists of 22,709 scaffolds with a total length of 945 bp with contig N50 of 116.6 kb. Using BRAKER2, we annotated a total of 30,068 genes. Analysis of the repetitive regions of the genome reveals the genome to contain 29.1% transposable elements, and the longnose gar to be the only other known vertebrate outside of the spotted gar to contain CR1, L2, Rex1, and Babar. These results highlight the potential utility of holostean genomes for understanding the evolution of vertebrate repetitive elements and provide a critical reference for comparative genomic studies utilizing ray-finned fish models. SignificanceOver half of all living vertebrates are teleost fishes, including numerous experimental models such as zebrafish (Danio rerio) and medaka (Oryzias latipes). However, translating research in teleost models to other organisms such as humans is often challenged by the fact that teleosts experienced a genome duplication event in their early evolutionary history. Recent genome sequencing of three holosteans, the sister lineage to teleosts that did not experience a genome duplication event, has revealed these taxa to be critical for linking homologs between teleosts and other vertebrates. Sequencing of holostean genomes remains limited, thereby impeding further comparative genomic studies. Here we fill this sampling gap through the genomic sequencing of the longnose gar (Lepisosteus osseus). This annotated reference genome will provide a useful resource for a range of comparative genomic applications that span fields as diverse as immunogenetics, developmental biology, and the understanding of regulatory sequence evolution.

genomics↗

bioRxiv2025

Nuclear genome profiling of two Mexican orchids of the genus Epidendrum

Characterizing genomic properties such as genome size, ploidy level, heterozygosity, and repetitive DNA proportion and composition without relying on genome assembly is crucial for profiling the genomes of non-model species. Little is known about the nuclear genome of the large neotropical orchid genus Epidendrum. This study compares genome profiles of Epidendrum anisatum and Epidendrum marmoratum, using flow cytometry and k-mer analysis approaches, as well as bioinformatics ploidy level estimation and repeatome characterization. Multiple depths of coverage, k values, and k-mer-based tools for genome size estimation were explored and contrasted with cytometry genome size estimations. Cytometry and k-mer analyses yielded a consistently higher genome size for E. anisatum (mean 1C genome size = 2.59 Gb) than E. marmoratum (mean 1C genome size = 1.13 Gb), which represents a 2.3-fold genome size difference. Both species were identified as diploid with no evidence of strict partial endoreplication. The most important aspects to be taken into account to improve genome size estimation were heterozygosity, depth of coverage, and the maximum k-mer coverage. The genomes of both species were found to be highly repetitive (63-73%) and heavily dominated by Ty3-gypsy retrotransposons, particularly those of the Ogre family. Additionally, the genome of E. anisatum was characterized by the presence of a 172 bp satellite (AniS1), which represented 11% of the genome size. Together, both Ty3-gypsy transposons and AniS1 shape the genome size difference between the two genomes. This study provides the first genome profiling for species in the genus Epidendrum, but also highlights the importance of using flow cytometry, cytogenetic approaches and bioinformatics techniques in combination for genome profiling.

genomics↗

bioRxiv2017

Parallel Altitudinal Clines Reveal Adaptive Evolution Of Genome Size In Zea mays

While the vast majority of genome size variation in plants is due to differences in repetitive sequence, we know little about how selection acts on repeat content in natural populations. Here we investigate parallel changes in intraspecific genome size and repeat content of domesticated maize (Zea mays) landraces and their wild relative teosinte across altitudinal gradients in Mesoamerica and South America. We combine genotyping, low coverage whole-genome sequence data, and flow cytometry to test for evidence of selection on genome size and individual repeat abundance. We find that population structure alone cannot explain the observed variation, implying that clinal patterns of genome size are maintained by natural selection. Our modeling additionally provides evidence of selection on individual heterochromatic knob repeats, likely due to their large individual contribution to genome size. To better understand the phenotypes driving selection on genome size, we conducted a growth chamber experiment using a population of highland teosinte exhibiting extensive variation in genome size. We find weak support for a positive correlation between genome size and cell size, but stronger support for a negative correlation between genome size and the rate of cell production. Reanalyzing published data of cell counts in maize shoot apical meristems, we then identify a negative correlation between cell production rate and flowering time. Together, our data suggest a model in which variation in genome size is driven by natural selection on flowering time across altitudinal clines, connecting intraspecific variation in repetitive sequence to important differences in adaptive phenotypes.\n\nAuthor summaryGenome size in plants can vary by orders of magnitude, but this variation has long been considered to be of little to no functional consequence. Studying three independent adaptations to high altitude in Zea mays, we find that genome size experiences parallel pressures from natural selection, causing a linear reduction in genome size with increasing altitude. Though reductions in repetitive content are responsible for the genome size change, we find that only those individual loci contributing most to the variation in genome size are individually targeted by selection. To identify the phenotype influenced by genome size, we study how variation in genome size within a single teosinte population impacts leaf growth and cell division. We find that genome size variation correlates negatively with the rate of cell division, suggesting that individuals with larger genomes require longer to complete a mitotic cycle. Finally, we reanalyze data from maize inbreds to show that faster cell division is correlated with earlier flowering, connecting observed variation in genome size to an important adaptive phenotype.

evolutionary biology↗

bioRxiv2019

Endogenization and excision of human herpesvirus 6 in human genomes

The genome of human herpesvirus 6 (HHV-6) is integrated within the nuclear genome of about 1% of humans, but how this came about is not clear. HHV-6 integrates into telomeres, and this has recently been associated with polymorphisms affecting MOV10L1. MOV10L1 is located on the subtelomere of chromosome 22q (chr22q) and is required to make PIWI-interacting RNAs (piRNAs). piRNAs block integration of transposons in the germline, so piRNA-mediated repression of HHV-6 integration has been suspected. Whether integrated HHV-6 can reactive into an infectious virus is also uncertain. In vitro, recombination of the viral genome along its terminal direct repeats (DRs) leads to excision from the telomere and viral reactivation, but the expected single DR "scar" has not been described in vivo. We analyzed whole-genome sequencing (WGS) data from 13,040 subjects, including 7,485 from Japan. We found an association between integrated HHV-6 and polymorphisms on chr22q in Japanese subjects. However, association with the reported MOV10L1 polymorphism was driven by physical linkage to a single ancient endogenous HHV-6A variant integrated into the telomere of chr22q in East Asians. We resolved the junction of the human chromosome with this viral genome using long read sequencing. Unexpectedly, an HHV-6B variant has also endogenized in chr22q; two endogenous HHV-6 variants at this locus thus account for 72% of all integrated HHV-6 in Japan. We also report human genomes carrying only one portion of the HHV-6B genome, a single DR, supporting in vivo excision and viral reactivation. Using WGS data from North American families, we show that the incidence of HHV-6 integration into the germline is lower than its prevalence, and that integrated HHV-6 is not associated with the reported variant in MOV10L1. Together these results explain the recently reported association between integrated HHV-6 and MOV10L1/piRNAs, suggest exaptation of HHV-6 in its coevolution with human chr22q, and clarify the evolution and risk of reactivation of the only intact non-retroviral genome known to be present in human germlines. SIGNIFICANCE STATEMENT Human herpesvirus 6 (HHV-6) infects most people during childhood, usually only causing fever and rash. Reactivation of HHV-6 has been linked to a number of neurological diseases including encephalitis, Alzheimers disease and multiple sclerosis. However, about 1% of people are born with the HHV-6 genome present within their genome, included in the end "cap" of one of their 46 chromosomes. Little is known about how and when HHV-6 genomes entered human genomes, whether or not they still do, and whether or not this poses risk for virus reactivation. We looked for HHV-6 in genome sequences from over 13,000 people. Most HHV-6 variants present in human genomes have been co-evolving with human chromosomes for many generations, and new integration events are rare. Surprisingly, in almost three fourths of Japanese people with HHV-6 in their genome, HHV-6 integrated in the same end of the same chromosome - 22q. Persistence of the HHV-6 genome within the short "cap" that preserves the end of chromosome 22q suggests that the integrated viral sequence may have taken on a useful function for this chromosome. We also found that some human genomes harbor only one part of the HHV-6 genome. This part is the same part that remains after experimental viral reactivation, during which most of the virus is cut out of the genome. This warrants assessment of the risk that integration of HHV-6 into inherited human genomes is not irreversible, and possibly leads to production of infectious virus.

genetics↗