BUSCO Applications from Quality Assessments to Gene Prediction and Phylogenomics.

Genomics promises comprehensive surveying of genomes and metagenomes, but rapidly changing technologies and expanding data volumes make evaluation of completeness a challenging task. Technical sequencing quality metrics can be complemented by quantifying completeness of genomic data sets in terms of the expected gene content of Benchmarking Universal Single-Copy Orthologs (BUSCO, http://busco.ezlab.org). The latest software release implements a complete refactoring of the code to make it more flexible and extendable to facilitate high-throughput assessments. The original six lineage assessment data sets have been updated with improved species sampling, 34 new subsets have been built for vertebrates, arthropods, fungi, and prokaryotes that greatly enhance resolution, and data sets are now also available for nematodes, protists, and plants. Here, we present BUSCO v3 with example analyses that highlight the wide-ranging utility of BUSCO assessments, which extend beyond quality control of genomics data sets to applications in comparative genomics analyses, gene predictor training, metagenomics, and phylogenomics

Advancing Genomics with OrthoDB, BUSCO, and the LEM Framework

The rapid growth of genomics data necessitates continuous advancements in bioinformatics tools. This presentation highlights the latest updates to our toolbox, including OrthoDB v11, BUSCO v5, and the LEM benchmarking framework. OrthoDB (https://www.orthodb.org) is a leading resource for gene orthology and functional annotations across diverse eukaryotes, prokaryotes, and viruses. Orthology facilitates precise bridging of gene function knowledge within the genomics sphere. OrthoDB v11 encompasses over 100 million genes from 18,000 prokaryotes and nearly 2,000 eukaryotes, providing extensive species coverage. The open-source OrthoLoger software (https://orthologer.ezlab.org) allows mapping of novel gene sets to precomputed orthologs, linking them to relevant annotations. BUSCO (https://busco.ezlab.org) serves as a standard tool for assessing the completeness of genome assemblies, transcriptomes, and predicted gene sets, complementing assembly contiguity measures like N50 values. A spin-off of OrthoDB, BUSCO evaluates the presence and coverage of marker genes, offering an evolutionarily-grounded expectation of gene content completeness. BUSCO v5 now automatically selects the most suitable dataset for evaluation, outperforming the popular CheckM tool. Its efficiency is particularly evident in large eukaryotic genomes, and it is uniquely capable of assessing both eukaryotic and prokaryotic species, making it applicable to metagenome-assembled genomes of unknown origin. The LEMMI (https://lemmi.ezlab.org) benchmarking framework, now in version 2, facilitates informed software tool selection. This Live Evaluation of Methods (LEM) for Metagenome Investigation uses a container-based approach for continuous benchmarking and effective end-user distribution. The versatile framework can be extended to other procedures, such as gene orthology inference with LEMOrtho (https://lemortho.ezlab.org). The LEM benchmarking approach aims to become a community-driven effort, allowing developers to showcase novel methods and users to access standardized, easy-to-use software. We encourage researchers to apply this framework in their domain and welcome feedback.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202

Protist taxonomic and functional diversity in soil, freshwater and marine ecosystems

Protists dominate eukaryotic diversity and play key functional roles in all ecosystems, particularly by catalyzing carbon and nutrient cycling. To date, however, a comparative analysis of their taxonomic and functional diversity that compares the major ecosystems on Earth (soil, freshwater and marine systems) is missing. Here, we present a comparison of protist diversity based on standardized high throughput 18S rRNA gene sequencing of soil, freshwater and marine environmental DNA. Soil and freshwater protist communities were more similar to each other than to marine protist communities, with virtually no overlap of Operational Taxonomic Units (OTUs) between terrestrial and marine habitats. Soil protists showed higher γ diversity than aquatic samples. Differences in taxonomic composition of the communities led to changes in a functional diversity among ecosystems, as expressed in relative abundance of consumers, phototrophs and parasites. Phototrophs (eukaryotic algae) dominated freshwater systems (49% of the sequences) and consumers soil and marine ecosystems (59% and 48%, respectively). The individual functional groups were composed of ecosystem- specific taxonomic groups. Parasites were equally common in all ecosystems, yet, terrestrial systems hosted more OTUs assigned to parasites of macro-organisms while aquatic systems contained mostly microbial parasitoids. Together, we show biogeographic patterns of protist diversity across major ecosystems on Earth, preparing the way for more focused studies that will help understanding the multiple roles of protists in the biosphere

Protist taxonomic and functional diversity in soil, freshwater and marine ecosystems

Protists dominate eukaryotic diversity and play key functional roles in all ecosystems, particularly by catalyzing carbon and nutrient cycling. To date, however, a comparative analysis of their taxonomic and functional diversity that compares the major ecosystems on Earth (soil, freshwater and marine systems) is missing. Here, we present a comparison of protist diversity based on standardized high throughput 18S rRNA gene sequencing of soil, freshwater and marine environmental DNA. Soil and freshwater protist communities were more similar to each other than to marine protist communities, with virtually no overlap of Operational Taxonomic Units (OTUs) between terrestrial and marine habitats. Soil protists showed higher γ diversity than aquatic samples. Differences in taxonomic composition of the communities led to changes in a functional diversity among ecosystems, as expressed in relative abundance of consumers, phototrophs and parasites. Phototrophs (eukaryotic algae) dominated freshwater systems (49% of the sequences) and consumers soil and marine ecosystems (59% and 48%, respectively). The individual functional groups were composed of ecosystem- specific taxonomic groups. Parasites were equally common in all ecosystems, yet, terrestrial systems hosted more OTUs assigned to parasites of macro-organisms while aquatic systems contained mostly microbial parasitoids. Together, we show biogeographic patterns of protist diversity across major ecosystems on Earth, preparing the way for more focused studies that will help understanding the multiple roles of protists in the biosphere

Genomic signatures accompanying the dietary shift to phytophagy in polyphagan beetles.

The diversity and evolutionary success of beetles (Coleoptera) are proposed to be related to the diversity of plants on which they feed. Indeed, the largest beetle suborder, Polyphaga, mostly includes plant eaters among its approximately 315,000 species. In particular, plants defend themselves with a diversity of specialized toxic chemicals. These may impose selective pressures that drive genomic diversification and speciation in phytophagous beetles. However, evidence of changes in beetle gene repertoires driven by such interactions remains largely anecdotal and without explicit hypothesis testing. We explore the genomic consequences of beetle-plant trophic interactions by performing comparative gene family analyses across 18 species representative of the two most species-rich beetle suborders. We contrast the gene contents of species from the mostly plant-eating suborder Polyphaga with those of the mainly predatory Adephaga. We find gene repertoire evolution to be more dynamic, with significantly more adaptive lineage-specific expansions, in the more speciose Polyphaga. Testing the specific hypothesis of adaptation to plant feeding, we identify families of enzymes putatively involved in beetle-plant interactions that underwent adaptive expansions in Polyphaga. There is notable support for the selection hypothesis on large gene families for glutathione S-transferase and carboxylesterase detoxification enzymes. Our explicit modeling of the evolution of gene repertoires across 18 species identifies putative adaptive lineage-specific gene family expansions that accompany the dietary shift towards plants in beetles. These genomic signatures support the popular hypothesis of a key role for interactions with plant chemical defenses, and for plant feeding in general, in driving beetle diversification

The Genome of the Toluene-Degrading Pseudomonas veronii Strain 1YdBTEX2 and Its Differential Gene Expression in Contaminated Sand.

The natural restoration of soils polluted by aromatic hydrocarbons such as benzene, toluene, ethylbenzene and m- and p-xylene (BTEX) may be accelerated by inoculation of specific biodegraders (bioaugmentation). Bioaugmentation mainly involves introducing bacteria that deploy their metabolic properties and adaptation potential to survive and propagate in the contaminated environment by degrading the pollutant. In order to better understand the adaptive response of cells during a transition to contaminated material, we analyzed here the genome and short-term (1 h) changes in genome-wide gene expression of the BTEX-degrading bacterium Pseudomonas veronii 1YdBTEX2 in non-sterile soil and liquid medium, both in presence or absence of toluene. We obtained a gapless genome sequence of P. veronii 1YdBTEX2 covering three individual replicons with a total size of 8 Mb, two of which are largely unrelated to current known bacterial replicons. One-hour exposure to toluene, both in soil and liquid, triggered massive transcription (up to 208-fold induction) of multiple gene clusters, such as toluene degradation pathway(s), chemotaxis and toluene efflux pumps. This clearly underlines their key role in the adaptive response to toluene. In comparison to liquid medium, cells in soil drastically changed expression of genes involved in membrane functioning (e.g., lipid composition, lipid metabolism, cell fatty acid synthesis), osmotic stress response (e.g., polyamine or trehalose synthesis, uptake of potassium) and putrescine metabolism, highlighting the immediate response mechanisms of P. veronii 1YdBTEX2 for successful establishment in polluted soil

A combined microbial and biogeochemical dataset from high-latitude ecosystems with respect to methane cycle.

High latitudes are experiencing intense ecosystem changes with climate warming. The underlying methane (CH4) cycling dynamics remain unresolved, despite its crucial climatic feedback. Atmospheric CH4 emissions are heterogeneous, resulting from local geochemical drivers, global climatic factors, and microbial production/consumption balance. Holistic studies are mandatory to capture CH4 cycling complexity. Here, we report a large set of integrated microbial and biogeochemical data from 387 samples, using a concerted sampling strategy and experimental protocols. The study followed international standards to ensure inter-comparisons of data amongst three high-latitude regions: Alaska, Siberia, and Patagonia. The dataset encompasses diferent representative environmental features (e.g. lake, wetland, tundra, forest soil) of these high-latitude sites and their respective heterogeneity (e.g. characteristic microtopographic patterns). The data included physicochemical parameters, greenhouse gas concentrations and emissions, organic matter characterization, trace elements and nutrients, isotopes, microbial quantifcation and composition. This dataset addresses the need for a robust physicochemical framework to conduct and contextualize future research on the interactions between climate change, biogeochemical cycles and microbial communities at highlatitudes

Acapsular Staphylococcus aureus with a non-functional agr regains capsule expression after passage through the bloodstream in a bacteremia mouse model

Selection pressures exerted on Staphylococcus aureus by host factors during infection may lead to the emergence of regulatory phenotypes better adapted to the infection site. Traits convenient for persistence may be fixed by mutation thus turning these mutants into microevolution endpoints. The feasibility that stable, non-encapsulated S. aureus mutants can regain expression of key virulence factors for survival in the bloodstream was investigated. S. aureus agr mutant HU-14 (IS256 insertion in agrC) from a patient with chronic osteomyelitis was passed through the bloodstream using a bacteriemia mouse model and derivative P3.1 was obtained. Although IS256 remained inserted in agrC, P3.1 regained production of capsular polysaccharide type 5 (CP5) and staphyloxanthin. Furthermore, P3.1 expressed higher levels of asp23/SigB when compared with parental strain HU-14. Strain P3.1 displayed decreased osteoclastogenesis capacity, thus indicating decreased adaptability to bone compared with strain HU-14 and exhibited a trend to be more virulent than parental strain HU-14. Strain P3.1 exhibited the loss of one IS256 copy, which was originally located in the HU-14 noncoding region between dnaG (DNA primase) and rpoD (sigA). This loss may be associated with the observed phenotype change but the mechanism remains unknown. In conclusion, S. aureus organisms that escape the infected bone may recover the expression of key virulence factors through a rapid microevolution pathway involving SigB regulation of key virulence factors.Fil: Suligoy Lozano, Carlos Mauricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Díaz, Rocío E.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Gehrke, Ana-katharina Elsa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Maimónides. Área de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico; ArgentinaFil: Ring, Natalie. University of Edinburgh; Reino UnidoFil: Yebra, Gonzalo. University of Edinburgh; Reino UnidoFil: Alves, Joana. University of Edinburgh; Reino UnidoFil: Gómez, Marisa Ileana. Universidad Maimónides. Área de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico; ArgentinaFil: Wendler, Sindy. Universitätsklinikum Jena Und Medizinische Fakultät; AlemaniaFil: Fitzgerald, J. Ross. University of Edinburgh; Reino UnidoFil: Tuchscherr, Lorena. Jena University Hospital; AlemaniaFil: Löffler, Bettina. Jena University Hospital; AlemaniaFil: Sordelli, Daniel Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Noto Llana, Mariangeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Buzzola, Fernanda Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentin

Comparative analysis of bones, mites, soil chemistry, nematodes and soil micro-Eukaryotes from a suspected homicide to estimate the post-mortem interval

Criminal investigations of suspected murder cases require estimating the post-mortem interval (PMI, or time after death) which is challenging for longer periods. Here we present the case of human remains found in a Swiss forest. We have used a multidisciplinary approach involving the analysis of bones, soil chemical characteristics, mites and nematodes (by microscopy) and micro-Eukaryotes (by Illumina high throughput sequencing). We analysed soil samples collected beneath the remains of the head, upper and lower body and “control” samples taken a few meters away. The PMI estimated on hair 14C-data via bomb peak radiocarbon dating gave a time range of 1 to 2 years before the finding of the remains on site. Cluster analyses for chemical constituents, nematodes, mites and micro- Eukaryotes revealed two clusters 1) head and upper body and 2) lower body and controls. From mite evidence, we conclude that the body was likely to have been brought to the site after death. However, chemical analyses, nematode community analyses and the analyses of micro-Eukaryotes indicate that decomposition took place at least partly on site. This study illustrates the usefulness of combining several lines of evidence for the study of homicide cases to better calibrate PMI inference tools

Soil protistology rebooted: 30 fundamental questions to start with

Protists are the most diverse eukaryotes. These microbes are keystone organisms of soil ecosystems and regulate essential processes of soil fertility such as nutrient cycling and plant growth. Despite this, protists have received little scientific attention, especially compared to bacteria, fungi and nematodes in soil studies. Recent methodological advances, particularly in molecular biology techniques, have made the study of soil protists more accessible, and have created a resurgence of interest in soil protistology. This ongoing revolution now enables comprehensive investigations of the structure and functioning of soil protist communities, paving the way to a new era in soil biology. Instead of providing an exhaustive review, we provide a synthesis of research gaps that should be prioritized in future studies of soil protistology to guide this rapidly developing research area. Based on a synthesis of expert opinion we propose 30 key questions covering a broad range of topics including evolution, phylogenetics, functional ecology, macroecology, paleoecology, and methodologies. These questions highlight a diversity of topics that will establish soil protistology as a hub discipline connecting different fundamental and applied fields such as ecology, biogeography, evolution, plant-microbe interactions, agronomy, and conservation biology. We are convinced that soil protistology has the potential to be one of the most exciting frontiers in biology