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GenomeDepot: data management system for microbial comparative genomics

Summary GenomeDepot is an open-source web-based platform for annotation, management, and comparative analysis of microbial genomic sequences and associated data including ortholog families, protein domains, operons, regulatory interactions, strain taxonomy, and sample metadata. GenomeDepot supports rapid creation of websites for user-defined genome collections that include bioinformatic tools for interactive genome browsing, Basic Local Alignment Search Tool (BLAST) search, annotation search, comparative genomic neighborhood visualization, and sequence download. Gene function annotations are generated by a customizable annotation pipeline. The pipeline runs annotation tools in Conda environments and can be easily extended with additional user-specified tools. Availability and implementation GenomeDepot is open source and distributed under the GNU General Public License via GitHub (https://github.com/aekazakov/genome-depot). GenomeDepot is implemented in Python and was tested in Ubuntu Linux. Full installation instructions and documentation are available at https://aekazakov.github.io/genome-depot/. GenomeDepot demo server is freely accessible at https://iseq.lbl.gov/demogd/.

Kazakov, Alexey [Lawrence Berkeley National Labora↗

VPF-Class: taxonomic assignment and host prediction of uncultivated viruses based on viral protein families

Abstract Motivation Two key steps in the analysis of uncultured viruses recovered from metagenomes are the taxonomic classification of the viral sequences and the identification of putative host(s). Both steps rely mainly on the assignment of viral proteins to orthologs in cultivated viruses. Viral Protein Families (VPFs) can be used for the robust identification of new viral sequences in large metagenomics datasets. Despite the importance of VPF information for viral discovery, VPFs have not yet been explored for determining viral taxonomy and host targets. Results In this work, we classified the set of VPFs from the IMG/VR database and developed VPF-Class. VPF-Class is a tool that automates the taxonomic classification and host prediction of viral contigs based on the assignment of their proteins to a set of classified VPFs. Applying VPF-Class on 731K uncultivated virus contigs from the IMG/VR database, we were able to classify 363K contigs at the genus level and predict the host of over 461K contigs. In the RefSeq database, VPF-class reported an accuracy of nearly 100% to classify dsDNA, ssDNA and retroviruses, at the genus level, considering a membership ratio and a confidence score of 0.2. The accuracy in host prediction was 86.4%, also at the genus level, considering a membership ratio of 0.3 and a confidence score of 0.5. And, in the prophages dataset, the accuracy in host prediction was 86% considering a membership ratio of 0.6 and a confidence score of 0.8. Moreover, from the Global Ocean Virome dataset, over 817K viral contigs out of 1 million were classified. Availability and implementation The implementation of VPF-Class can be downloaded from https://github.com/biocom-uib/vpf-tools. Supplementary information Supplementary data are available at Bioinformatics online.

59 BASIC BIOLOGICAL SCIENCES↗

Meta2DB: Curated Shotgun Metagenomic Feature Sets and Metadata for Health State Prediction

Meta2DB is a curated metagenomic and metadata database that provides structurally consistent microbiome taxonomy feature count tables for 13 897 samples across 84 studies, 23 disease states, and 34 geographical locations. All samples were uniformly processed using a streamlined metagenomic classification pipeline that employs a unique and comprehensive reference database indexed to contain all sequences across all kingdoms of life that were present in the NCBI Nucleotide (nt) database retrieved on 4 January 2023. This pipeline leverages high-performance computing (HPC) resources at Lawrence Livermore National Laboratory and was used to process 50TB of publicly available raw metagenomic sequence data. Extensive metadata curation was carried out through a combination of manual curation and automated parsing, producing a consistent inter-study metadata table specifically structured to facilitate training of ML models for prediction of human health.

Kok, C [Lawrence Livermore National Laboratory (LL↗

The microbial rare biosphere: current concepts, methods and ecological principles

ABSTRACT Our ability to describe the highly diverse pool of low abundance populations present in natural microbial communities is increasing at an unprecedented pace. Yet we currently lack an integrative view of the key taxa, functions and metabolic activity which make-up this communal pool, usually referred to as the ‘rare biosphere’, across the domains of life. In this context, this review examines the microbial rare biosphere in its broader sense, providing an historical perspective on representative studies which enabled to bridge the concept from macroecology to microbial ecology. It then addresses our current knowledge of the prokaryotic rare biosphere, and covers emerging insights into the ecology, taxonomy and evolution of low abundance microeukaryotic, viral and host-associated communities. We also review recent methodological advances and provide a synthetic overview on how the rare biosphere fits into different conceptual models used to explain microbial community assembly mechanisms, composition and function.

Pascoal, Francisco↗

Identification of 2 Novel Subtypes of Hepatitis C Virus Genotype 8 and a Potential New Genotype Successfully Treated With Direct Acting Antivirals

Abstract Background Hepatitis C virus (HCV) has high genetic diversity and is classified into 8 genotypes and >90 subtypes, with some endemic to specific world regions. This could compromise direct-acting antiviral efficacy and global HCV elimination. Methods We characterized HCV subtypes “rare” in the United Kingdom (non-1a/1b/2b/3a/4d) by means of whole-genome sequencing via a national surveillance program. Genetic analyses to determine the genotype of samples with unresolved genotypes were undertaken by comparison with International Committee on Taxonomy of Viruses HCV reference sequences. Results Two HCV variants were characterized as being closely related to the recently identified genotype (GT) 8, with >85% pairwise genetic distance similarity to GT8 sequences and within the typical intersubtype genetic distance range. The individuals infected by the variants were UK residents originally from Pakistan and India. In contrast, a third variant was only confidently identified to be more similar to GT6 compared with other genotypes across 6% of the genome and was isolated from a UK resident originally from Guyana. All 3 were cured with pangenotypic direct-acting antivirals (sofosbuvir-velpatasvir or glecaprevir-pibrentasvir) despite the presence of resistance polymorphisms in NS3 (80K/168E), NS5A (28V/30S/62L/92S/93S) and NS5B (159F). Conclusions This study expands our knowledge of HCV diversity by identifying 2 new GT8 subtypes and potentially a new genotype.

59 BASIC BIOLOGICAL SCIENCES↗

Disentangling the effects of sulfate and other seawater ions on microbial communities and greenhouse gas emissions in a coastal forested wetland

Seawater intrusion into freshwater wetlands causes changes in microbial communities and biogeochemistry, but the exact mechanisms driving these changes remain unclear. Here we use a manipulative laboratory microcosm experiment, combined with DNA sequencing and biogeochemical measurements, to tease apart the effects of sulfate from other seawater ions. We examined changes in microbial taxonomy and function as well as emissions of carbon dioxide, methane, and nitrous oxide in response to changes in ion concentrations. Greenhouse gas emissions and microbial richness and composition were altered by artificial seawater regardless of whether sulfate was present, whereas sulfate alone did not alter emissions or communities. Surprisingly, addition of sulfate alone did not lead to increases in the abundance of sulfate reducing bacteria or sulfur cycling genes. Similarly, genes involved in carbon, nitrogen, and phosphorus cycling responded more strongly to artificial seawater than to sulfate. These results suggest that other ions present in seawater, not sulfate, drive ecological and biogeochemical responses to seawater intrusion and may be drivers of increased methane emissions in soils that received artificial seawater addition. A better understanding of how the different components of salt water alter microbial community composition and function is necessary to forecast the consequences of coastal wetland salinization.

54 ENVIRONMENTAL SCIENCES↗

IMG/VR v4: an expanded database of uncultivated virus genomes within a framework of extensive functional, taxonomic, and ecological metadata

Viruses are widely recognized as critical members of all microbiomes. Metagenomics enables large-scale exploration of the global virosphere, progressively revealing the extensive genomic diversity of viruses on Earth and highlighting the myriad of ways by which viruses impact biological processes. IMG/VR provides access to the largest collection of viral sequences obtained from (meta)genomes, along with functional annotation and rich metadata. A web interface enables users to efficiently browse and search viruses based on genome features and/or sequence similarity. Here, for this work, we present the fourth version of IMG/VR, composed of >15 million virus genomes and genome fragments, a ≈6-fold increase in size compared to the previous version. These clustered into 8.7 million viral operational taxonomic units, including 231 408 with at least one high-quality representative. Viral sequences in IMG/VR are now systematically identified from genomes, metagenomes, and metatranscriptomes using a new detection approach (geNomad), and IMG standard annotation are complemented with genome quality estimation using CheckV, taxonomic classification reflecting the latest taxonomic standards, and microbial host taxonomy prediction. IMG/VR v4 is available at https://img.jgi.doe.gov/vr, and the underlying data are available to download at https://genome.jgi.doe.gov/portal/IMG_VR.

59 BASIC BIOLOGICAL SCIENCES↗

NMPFamsDB: a database of novel protein families from microbial metagenomes and metatranscriptomes

Abstract The Novel Metagenome Protein Families Database (NMPFamsDB) is a database of metagenome- and metatranscriptome-derived protein families, whose members have no hits to proteins of reference genomes or Pfam domains. Each protein family is accompanied by multiple sequence alignments, Hidden Markov Models, taxonomic information, ecosystem and geolocation metadata, sequence and structure predictions, as well as 3D structure models predicted with AlphaFold2. In its current version, NMPFamsDB hosts over 100 000 protein families, each with at least 100 members. The reported protein families significantly expand (more than double) the number of known protein sequence clusters from reference genomes and reveal new insights into their habitat distribution, origins, functions and taxonomy. We expect NMPFamsDB to be a valuable resource for microbial proteome-wide analyses and for further discovery and characterization of novel functions. NMPFamsDB is publicly available in http://www.nmpfamsdb.org/ or https://bib.fleming.gr/NMPFamsDB.

59 BASIC BIOLOGICAL SCIENCES↗

IMG/PR: a database of plasmids from genomes and metagenomes with rich annotations and metadata

Plasmids are mobile genetic elements found in many clades of Archaea and Bacteria. They drive horizontal gene transfer, impacting ecological and evolutionary processes within microbial communities, and hold substantial importance in human health and biotechnology. To support plasmid research and provide scientists with data of an unprecedented diversity of plasmid sequences, we introduce the IMG/PR database, a new resource encompassing 699 973 plasmid sequences derived from genomes, metagenomes and metatranscriptomes. IMG/PR is the first database to provide data of plasmid that were systematically identified from diverse microbiome samples. IMG/PR plasmids are associated with rich metadata that includes geographical and ecosystem information, host taxonomy, similarity to other plasmids, functional annotation, presence of genes involved in conjugation and antibiotic resistance. The database offers diverse methods for exploring its extensive plasmid collection, enabling users to navigate plasmids through metadata-centric queries, plasmid comparisons and BLAST searches. The web interface for IMG/PR is accessible at https://img.jgi.doe.gov/pr. Plasmid metadata and sequences can be downloaded from https://genome.jgi.doe.gov/portal/IMG_PR.

59 BASIC BIOLOGICAL SCIENCES↗

VirJenDB: a FAIR (meta)data and bioinformatics platform for all viruses

High-throughput sequencing has generated an unprecedented volume of data. However, researcher-submitted data in repositories requires extensive curation and quality control for reuse. These tasks are hindered by the multiplicity of repositories, the sheer volume of the data, and the complexity of virus (meta)data curation. To address these challenges, VirJenDB offers a user-friendly platform to facilitate versioned, community-driven curation, and ontology development. Virus sequences were ingested from 16 sources, including ~200 fields of metadata or standards, covering taxonomy, sample, and host information. Up to 85 metadata fields have undergone at least one round of curation, and are linked to 15.4 million virus sequences, with 88 % from those infecting eukaryotes and the remaining infecting prokaryotes. Subsets were created, including a novel collection of 0.91 million viral operational taxonomic unit (vOTU) sequences across all viruses, while keeping the original sequences from each vOTU to facilitate downstream analyses, e.g. sequence variation. The VirJenDB web portal (https://www.virjendb.org) provides HTTPS and Application Programming Interface (API) access to the sequence datasets and metadata, offering a search engine, filtering, download, visualizations, and documentation. VirJenDB aims to connect the phage and eukaryotic virus research communities by supporting webtool integration, meta-analyses, and metadata schema extensions.

Saghaei, Shahram↗

Proposed minimal standards for description of methanogenic archaea

Methanogenic archaea are a diverse, polyphyletic group of strictly anaerobic prokaryotes capable of producing methane as their primary metabolic product. It has been over three decades since minimal standards for their taxonomic description have been proposed. In light of advancements in technology and amendments in systematic microbiology, revision of the older criteria for taxonomic description is essential. Most of the previously recommended minimum standards regarding phenotypic characterization of pure cultures are maintained. Electron microscopy and chemotaxonomic methods like whole-cell protein and lipid analysis are desirable but not required. Because of advancements in DNA sequencing technologies, obtaining a complete or draft whole genome sequence for type strains and its deposition in a public database are now mandatory. Genomic data should be used for rigorous comparison to close relatives using overall genome related indices such as average nucleotide identity and digital DNA–DNA hybridization. Phylogenetic analysis of the 16S rRNA gene is also required and can be supplemented by phylogenies of the mcrA gene and phylogenomic analysis using multiple conserved, single-copy marker genes. Additionally, it is now established that culture purity is not essential for studying prokaryotes, and description of Candidatus methanogenic taxa using single-cell or metagenomics along with other appropriate criteria is a viable alternative. The revisions to the minimal criteria proposed here by the members of the Subcommittee on the Taxonomy of Methanogenic Archaea of the International Committee on Systematics of Prokaryotes should allow for rigorous yet practical taxonomic description of these important and diverse microbes.

Microbiology↗

A comparative genome analysis of the Bacillota ( Firmicutes ) class Dehalobacteriia

Dehalobacterium formicoaceticum is recognized for its ability to anaerobically ferment dichloromethane (DCM), and a catabolic model has recently been proposed. D. formicoaceticum is currently the only axenic representative of its class, the Dehalobacteriia, according to the Genome Taxonomy Database. However, substantial additional diversity has been revealed in this lineage through culture-independent exploration of anoxic habitats. Here we performed a comparative analysis of 10 members of the Dehalobacteriia, representing three orders, and infer that anaerobic DCM degradation appears to be a recently acquired trait only present in some members of the order Dehalobacteriales. Inferred traits common to the class include the use of amino acids as carbon and energy sources for growth, energy generation via a remarkable range of putative electron-bifurcating protein complexes and the presence of S-layers. The ability of D. formicoaceticum to grow on serine without DCM was experimentally confirmed and a high abundance of the electron-bifurcating protein complexes and S-layer proteins was noted when this organism was grown on DCM. We suggest that members of the Dehalobacteriia are low-abundance fermentative scavengers in anoxic habitats.

59 BASIC BIOLOGICAL SCIENCES↗

Assessment of the Distributed Ledger Technology for Energy Sector Industrial and Operational Applications Using the MITRE ATT&CK® ICS Matrix

In recent times, Distributed Ledger Technology (DLT) has gained significant attention for its potential application in the energy sector. Utilizing blockchain and DLT has demonstrated the ability to enhance the resilience of the electric infrastructure, which will support a more flexible infrastructure and advance grid modernization. However, the deployment of these technologies increases the overall attack surface. The MITRE ATT&CK® matrices have been developed to document an adversary’s tactics and techniques based on real-world observations. The MITRE ATT&CK® matrices provide a common taxonomy for offense and defense and have become a valuable conceptual tool across multiple cybersecurity disciplines for conveying threat intelligence, performing testing through red teaming or adversary emulation, and enhancing network and system defenses against intrusions. The MITRE ATT&CK® for Industrial Control Systems (ICS) matrix was created to provide knowledge about adversary behavior in the ICS technology domain. This study analyzes the relevance of various tactics and techniques across a seven-layer DLT engineering and cybersecurity stack, known as the DLT stack, designed by the Cybersecurity Taskforce under IEEE P2418.5 - Standard for Blockchain in Energy working group sponsored by Power and Energy Systems - Smart Buildings, Loads and Customer Systems (PES/SBLC) Technical Committee. Additionally, this paper identifies specific mitigation strategies tailored to the energy ICS environment

42 ENGINEERING↗

Advancing Energy Equity Considerations in Distribution Systems Planning

Current distribution system planning (DSP) processes do not explicitly account for energy equity considerations, such as who is most affected by power system burdens, where those burdens are concentrated, and what investments can be made to improve baseline conditions. This paper proposes an iterative framework for advancing energy equity as an objective of the DSP process, showing how measurement strategies, or metrics (informed by conceptual foundations of energy justice), can be applied to benchmark equity performance at various stages. This methodology is applied for equity-aware distributed energy resource (DER) hosting capacity analysis and outage analysis to provide critical insights on infrastructure upgrade decisions compared to a business-as-usual (BAU) case. The analysis is performed on a taxonomy feeder representing the West Coast urban/semi-urban system with augmentation of electric vehicles (EVs) and rooftop solar photovoltaic (PV) generators. The study considers disadvantaged community (DAC) and non-disadvantaged community (NDAC) load regions to enable equity-aware simulations. The results demonstrate how equity-aware planning could reveal the limitations of the traditional DSP process as DAC regions are found to have lower DER hosting capacity and higher outage vulnerability. Overall, this work provides insights on the need to incorporate energy equity as an integral part of the DSP process.

Energy Equity, Distribution System Planning, DER a↗

Enhancing Smart Home Privacy: A Tutorial on Local Differential Privacy Techniques for Frequency and Mean Estimation

The ubiquity of Internet of Things (IoT) systems has seamlessly integrated into our daily lives, particularly in smart homes where devices continuously monitor and optimize our living environments. These systems significantly contribute to home automation, energy efficiency, and overall comfort. However, this widespread connectivity poses inherent risks linked to the streaming of sensitive household data, necessitating robust privacy preservation mechanisms. This tutorial systematically examines privacy preservation through local differential privacy (LDP), with a particular focus on frequency and mean estimation techniques for smart home applications. Here, we present a comprehensive taxonomy of smart home data formats and provide detailed implementation guidance for event-based and w-event LDP mechanisms. Through practical examples using smart thermostats and HVAC systems, we demonstrate how these techniques can be effectively deployed in real-world scenarios. The tutorial concludes by examining emerging research directions, including adaptive privacy budgets and federated learning approaches, establishing a foundation for privacy-preserving smart home deployments.

Kotevska, Olivera [Oak Ridge National Laboratory (↗

Feedback and Oscillations: Constructing Feedback Systems for Root Cause Analysis of Oscillations in Power Grids

Dynamic phenomena linked to inverter-based resources (IBRs) have gained global attention. Several IBR-induced dynamics have caused bulk power system-connected wind or solar power plants to trip, and some have even led to widespread outages. In addition, many oscillations have been observed involving IBR power plants. In 2023, the IEEE Power & Energy Society (PES) IBR Subsynchronous Oscillations (SSO) task force published a journal article, “Real-World Subsynchronous Oscillation Events in Power Grids With High Penetrations of Inverter-Based Resources,” in which 19 IBR oscillation events were examined for their causation. Earlier in 2020, another PES task force article, “Definition and Classification of Power System Stability-Revisited & Extended,” authored by prominent academics, introduced converter-driven stability as a new category of stability. The international power grid industry community also took action by publishing the CIGRE Green Book, Power System Dynamic Modelling and Analysis in Evolving Networks (led by Babak Badrzadeh and Zia Emin) in 2024. In August 2024, the Energy Systems Integration Group (ESIG) released a practical guide led by Nick Miller, “Diagnosis and Mitigation of Observed Oscillations in IBR-Dominant Power System: A Practical Guide.” The goal of the guide is to assist practicing engineers in making initial judgments and conducting detailed analyses about oscillations. Finally, when addressing the classification of stability and oscillations, the guide emphasizes a causality-based taxonomy for grouping, such as voltage control-induced oscillations, synchronization-induced oscillations, and frequency or active power control-induced oscillations.

Fan, Lingling [Univ. of South Florida, Tampa, FL (↗

Application of DLT cybersecurity stack to TES applications for a scalable, cybersecure, and interoperable future

Transactive Energy Systems (TES) are expected to improve upon existing grid operations and capabilities by enabling the integration of traditional grid resources with distributed energy resources (DER). Distributed Ledger Technology or DLT (e.g., blockchain) presents itself as a viable instrument to support decentralized, autonomous, and tamper-evident applications, which can be leveraged within TES's ecosystem. DLTs can provide pertinent security controls including access controls, data immutability, and traceability in addition to other well-known advantages such as decentralization and scalability. This work demonstrates the DLT Cybersecurity stack and its applicability to TES-based use-cases/applications. The seven-layer DLT cybersecurity stack is a DLT-agnostic framework that can quickly be used to classify and group the individual needs of an application into the different processing and cybersecurity layers offered by a DLT using a common taxonomy and an architectural mapping framework. This enables application engineers to demystify and strengthen the overall security aspects of their systems while maintaining an open perspective towards features and drawbacks that may hinder their performance in real-world scenarios. The paper leverages the work performed by the IEEE P2418.5 - Blockchain for Energy Standards working group.

Blockchain, blockchain interoperability, Cybersecu↗

Identifying Adversarial Cyber-Activity in Operational Technology Environments Using Bayesian Networks

Critical infrastructure and other operational technology (OT) environments face increasing cybersecurity risks from adversarial behavior. This paper describes the development of a risk model using a Bayesian network to enhance the comprehension of observable cyber events caused by malicious activity in OT environments. The core of the Bayesian network is a process model that describes the stages of adversary behavior. The remainder of the model is based on the MITRE ATT&CK® for Industrial Control Systems (ICS) taxonomy, which includes tactics and techniques that may be used by the adversary. The observables provide evidence for adversary behavior through the intermediary technique and tactic nodes. One challenge in constructing this model is a lack of open-source data from cyber-attacks on OT systems. This paper discusses learning from limited data, the elicitation of expert opinion to construct the conditional probability tables when data is scarce, and the refinement of the most difficult conditional probabilities tables using several forms of sensitivity analyses. Finally, the Bayesian network is demonstrated using two historical case studies: the DarkSide ransomware attack on the Colonial Pipeline and the destructive cyberattack targeting the ThyssenKrupp blast furnace. Index Terms—Cybersecurity, industrial control systems, operational technology

97 - MATHEMATICS AND COMPUTING↗