Metatranscriptomes of California grassland soil microbial communities in response to rewetting.

When very dry soil is rewet, rapid stimulation of microbial activity has important implications for ecosystem biogeochemistry, yet associated changes in microbial transcription are poorly known. Here, we present metatranscriptomes of California annual grassland soil microbial communities, collected over 1 week from soils rewet after a summer drought-providing a time series of short-term transcriptional response during rewetting.

Whole community shotgun metagenomes of two biological soil crust types from the Mojave Desert.

We present six whole community shotgun metagenomic sequencing data sets of two types of biological soil crusts sampled at the ecotone of the Mojave Desert and Colorado Desert in California. These data will help us understand the diversity and function of biocrust microbial communities, which are essential for desert ecosystems.

Metatranscriptomes of two biological soil crust types from the Mojave desert in response to wetting.

We present eight metatranscriptomic datasets of light algal and cyanolichen biological soil crusts from the Mojave Desert in response to wetting. These data will help us understand gene expression patterns in desert biocrust microbial communities after they have been reactivated by the addition of water.

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.

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

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.

Unraveling the functional dark matter through global metagenomics.

Metagenomes encode an enormous diversity of proteins, reflecting a multiplicity of functions and activities1,2. Exploration of this vast sequence space has been limited to a comparative analysis against reference microbial genomes and protein families derived from those genomes. Here, to examine the scale of yet untapped functional diversity beyond what is currently possible through the lens of reference genomes, we develop a computational approach to generate reference-free protein families from the sequence space in metagenomes. We analyse 26,931 metagenomes and identify 1.17 billion protein sequences longer than 35 amino acids with no similarity to any sequences from 102,491 reference genomes or the Pfam database3. Using massively parallel graph-based clustering, we group these proteins into 106,198 novel sequence clusters with more than 100 members, doubling the number of protein families obtained from the reference genomes clustered using the same approach. We annotate these families on the basis of their taxonomic, habitat, geographical and gene neighbourhood distributions and, where sufficient sequence diversity is available, predict protein three-dimensional models, revealing novel structures. Overall, our results uncover an enormously diverse functional space, highlighting the importance of further exploring the microbial functional dark matter.

Standardized naming of microbiome samples in Genomes OnLine Database.

The power of next-generation sequencing has resulted in an explosive growth in the number of projects aiming to understand the metagenomic diversity of complex microbial environments. The interdisciplinary nature of this microbiome research community, along with the absence of reporting standards for microbiome data and samples, poses a significant challenge for follow-up studies. Commonly used names of metagenomes and metatranscriptomes in public databases currently lack the essential information necessary to accurately describe and classify the underlying samples, which makes a comparative analysis difficult to conduct and often results in misclassified sequences in data repositories. The Genomes OnLine Database (GOLD) (https:// gold.jgi.doe.gov/) at the Department of Energy Joint Genome Institute has been at the forefront of addressing this challenge by developing a standardized nomenclature system for naming microbiome samples. GOLD, currently in its twenty-fifth anniversary, continues to enrich the research community with hundreds of thousands of metagenomes and metatranscriptomes with well-curated and easy-to-understand names. Through this manuscript, we describe the overall naming process that can be easily adopted by researchers worldwide. Additionally, we propose the use of this naming system as a best practice for the scientific community to facilitate better interoperability and reusability of microbiome data.

A Practical Curriculum Design and Learning Effectiveness Evaluation of Competence-Oriented Instruction Strategy Integration: A Case Study of Taiwan Skills-Based Senior High School.

The 12-Year Basic Education Curricula not only follow the objectives of previous curriculum syllabus development, but they place more focus on competence-oriented instruction, which aims to emphasize the importance of combining the curriculum with life situations that are not solely based on learning knowledge and skills. This study aims to investigate the results of the students' learning effectiveness and learning engagement after adding competence-oriented instructional strategies into electrical engineering practical subjects offered by the Department of Electrical Engineering in skills-based senior high schools, and, at the same time, to figure out the difference in learning effectiveness using different instructional strategies. Two classes of students from the Department of Electrical Engineering major in electrical engineering practical subjects in one skills-based senior high school in Central Taiwan were chosen as the participants for this study. By way of pre-test-post-test research design and heterogeneous grouping, a 10-week instruction experiment consisting of two groups and occurring over the course of 30 classes was conducted, wherein competence-oriented instructional strategies were used in an experimental group, while traditional didactic instructional strategies were used in a control group. By analyzing the collection of quantitative and qualitative data through competence-oriented instructional strategies, the instruction effectiveness and feasibility of the basic electricity practical curriculum were developed as the study topic for understanding how competence-oriented instruction can be implemented into the practical curriculum of the electrical engineering and electronic engineering group. According to the research objective, the results were concluded as follows: (1) Students of the Department of Electrical Engineering have a slightly better learning effectiveness in electrical engineering practice under competence-oriented instructional strategies compared with those who learned under the traditional didactic instructional strategies; (2) there is a significant difference in the students' learning engagement in electrical engineering practice from the Department of Electrical Engineering under competence-oriented instructional strategies compared with those who learned under the traditional didactic instructional strategies. The conclusion of this study emphasizes designing questions based on life situations, thereby applying what students have learned to solve problems they encounter in their daily lives. Compared with traditional didactic instructional strategies, competence-oriented instructional strategies not only have a better learning effectiveness and provide flexibility for the students to solve problems and provide analysis for situations, they also have broader applicability and an increased number of positive benefits when it comes to students' group interactions and sharing.

Twenty-five years of Genomes OnLine Database (GOLD): data updates and new features in v.9.

The Genomes OnLine Database (GOLD) (https://gold.jgi.doe.gov/) at the Department of Energy Joint Genome Institute (DOE-JGI) continues to maintain its role as one of the flagship genomic metadata repositories of the world. The ever-increasing number of projects and metadata are freely available to the user community world-wide. GOLD's metadata is consumed by scientists and remains an important source for large-scale comparative genomics analysis initiatives. Encouraged by this active user engagement and growth, GOLD has continued to add new components and capabilities. The new features such as a public Application Programming Interface (API) and Ecosystem landing page as well as the growth of different entities in this current GOLD v.9 edition are described in detail in this manuscript.

The IMG/M data management and analysis system v.7: content updates and new features.

The Integrated Microbial Genomes & Microbiomes system (IMG/M: https://img.jgi.doe.gov/m/) at the Department of Energy (DOE) Joint Genome Institute (JGI) continues to provide support for users to perform comparative analysis of isolate and single cell genomes, metagenomes, and metatranscriptomes. In addition to datasets produced by the JGI, IMG v.7 also includes datasets imported from public sources such as NCBI Genbank, SRA, and the DOE National Microbiome Data Collaborative (NMDC), or submitted by external users. In the past couple years, we have continued our effort to help the user community by improving the annotation pipeline, upgrading the contents with new reference database versions, and adding new analysis functionalities such as advanced scaffold search, Average Nucleotide Identity (ANI) for high-quality metagenome bins, new cassette search, improved gene neighborhood display, and improvements to metatranscriptome data display and analysis. We also extended the collaboration and integration efforts with other DOE-funded projects such as NMDC and DOE Biology Knowledgebase (KBase).

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, 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.

Expansion of the global RNA virome reveals diverse clades of bacteriophages.

High-throughput RNA sequencing offers broad opportunities to explore the Earth RNA virome. Mining 5,150 diverse metatranscriptomes uncovered >2.5 million RNA virus contigs. Analysis of >330,000 RNA-dependent RNA polymerases (RdRPs) shows that this expansion corresponds to a 5-fold increase of the known RNA virus diversity. Gene content analysis revealed multiple protein domains previously not found in RNA viruses and implicated in virus-host interactions. Extended RdRP phylogeny supports the monophyly of the five established phyla and reveals two putative additional bacteriophage phyla and numerous putative additional classes and orders. The dramatically expanded phylum Lenarviricota, consisting of bacterial and related eukaryotic viruses, now accounts for a third of the RNA virome. Identification of CRISPR spacer matches and bacteriolytic proteins suggests that subsets of picobirnaviruses and partitiviruses, previously associated with eukaryotes, infect prokaryotic hosts.

Draft Metagenome Sequences of the Sphagnum (Peat Moss) Microbiome from Ambient and Warmed Environments across Europe.

We present 49 metagenome assemblies of the microbiome associated with Sphagnum (peat moss) collected from ambient, artificially warmed, and geothermally warmed conditions across Europe. These data will enable further research regarding the impact of climate change on plant-microbe symbiosis, ecology, and ecosystem functioning of northern peatland ecosystems.

Sodalis ligni Strain 159R Isolated from an Anaerobic Lignin-Degrading Consortium.

Novel bacterial isolates with the capabilities of lignin depolymerization, catabolism, or both, could be pertinent to lignocellulosic biofuel applications. In this study, we aimed to identify anaerobic bacteria that could address the economic challenges faced with microbial-mediated biotechnologies, such as the need for aeration and mixing. Using a consortium seeded from temperate forest soil and enriched under anoxic conditions with organosolv lignin as the sole carbon source, we successfully isolated a novel bacterium, designated 159R. Based on the 16S rRNA gene, the isolate belongs to the genus Sodalis in the family Bruguierivoracaceae. Whole-genome sequencing revealed a genome size of 6.38 Mbp and a GC content of 55 mol%. To resolve the phylogenetic position of 159R, its phylogeny was reconstructed using (i) 16S rRNA genes of its closest relatives, (ii) multilocus sequence analysis (MLSA) of 100 genes, (iii) 49 clusters of orthologous groups (COG) domains, and (iv) 400 conserved proteins. Isolate 159R was closely related to the deadwood associated Sodalis guild rather than the tsetse fly and other insect endosymbiont guilds. Estimated genome-sequence-based digital DNA-DNA hybridization (dDDH), genome percentage of conserved proteins (POCP), and an alignment analysis between 159R and the Sodalis clade species further supported that isolate 159R was part of the Sodalis genus and a strain of Sodalis ligni. We proposed the name Sodalis ligni str. 159R (=DSM 110549 = ATCC TSD-177). IMPORTANCE Currently, in the paper industry, paper mill pulping relies on unsustainable and costly processes to remove lignin from lignocellulosic material. A greener approach is biopulping, which uses microbes and their enzymes to break down lignin. However, there are limitations to biopulping that prevent it from outcompeting other pulping processes, such as requiring constant aeration and mixing. Anaerobic bacteria are a promising alternative source for consolidated depolymerization of lignin and its conversion to valuable by-products. We presented Sodalis ligni str. 159R and its characteristics as another example of potential mechanisms that can be developed for lignocellulosic applications.

Expanding the genomic encyclopedia of Actinobacteria with 824 isolate reference genomes.

The phylum Actinobacteria includes important human pathogens like Mycobacterium tuberculosis and Corynebacterium diphtheriae and renowned producers of secondary metabolites of commercial interest, yet only a small part of its diversity is represented by sequenced genomes. Here, we present 824 actinobacterial isolate genomes in the context of a phylum-wide analysis of 6,700 genomes including public isolates and metagenome-assembled genomes (MAGs). We estimate that only 30%-50% of projected actinobacterial phylogenetic diversity possesses genomic representation via isolates and MAGs. A comparison of gene functions reveals novel determinants of host-microbe interaction as well as environment-specific adaptations such as potential antimicrobial peptides. We identify plasmids and prophages across isolates and uncover extensive prophage diversity structured mainly by host taxonomy. Analysis of >80,000 biosynthetic gene clusters reveals that horizontal gene transfer and gene loss shape secondary metabolite repertoire across taxa. Our observations illustrate the essential role of and need for high-quality isolate genome sequences.

Genomics, Exometabolomics, and Metabolic Probing Reveal Conserved Proteolytic Metabolism of Thermoflexus hugenholtzii and Three Candidate Species From China and Japan.

Thermoflexus hugenholtzii JAD2T, the only cultured representative of the Chloroflexota order Thermoflexales, is abundant in Great Boiling Spring (GBS), NV, United States, and close relatives inhabit geothermal systems globally. However, no defined medium exists for T. hugenholtzii JAD2T and no single carbon source is known to support its growth, leaving key knowledge gaps in its metabolism and nutritional needs. Here, we report comparative genomic analysis of the draft genome of T. hugenholtzii JAD2T and eight closely related metagenome-assembled genomes (MAGs) from geothermal sites in China, Japan, and the United States, representing "Candidatus Thermoflexus japonica," "Candidatus Thermoflexus tengchongensis," and "Candidatus Thermoflexus sinensis." Genomics was integrated with targeted exometabolomics and 13C metabolic probing of T. hugenholtzii. The Thermoflexus genomes each code for complete central carbon metabolic pathways and an unusually high abundance and diversity of peptidases, particularly Metallo- and Serine peptidase families, along with ABC transporters for peptides and some amino acids. The T. hugenholtzii JAD2T exometabolome provided evidence of extracellular proteolytic activity based on the accumulation of free amino acids. However, several neutral and polar amino acids appear not to be utilized, based on their accumulation in the medium and the lack of annotated transporters. Adenine and adenosine were scavenged, and thymine and nicotinic acid were released, suggesting interdependency with other organisms in situ. Metabolic probing of T. hugenholtzii JAD2T using 13C-labeled compounds provided evidence of oxidation of glucose, pyruvate, cysteine, and citrate, and functioning glycolytic, tricarboxylic acid (TCA), and oxidative pentose-phosphate pathways (PPPs). However, differential use of position-specific 13C-labeled compounds showed that glycolysis and the TCA cycle were uncoupled. Thus, despite the high abundance of Thermoflexus in sediments of some geothermal systems, they appear to be highly focused on chemoorganotrophy, particularly protein degradation, and may interact extensively with other microorganisms in situ.

DOE JGI Metagenome Workflow.

The DOE Joint Genome Institute (JGI) Metagenome Workflow performs metagenome data processing, including assembly; structural, functional, and taxonomic annotation; and binning of metagenomic data sets that are subsequently included into the Integrated Microbial Genomes and Microbiomes (IMG/M) (I.-M. A. Chen, K. Chu, K. Palaniappan, A. Ratner, et al., Nucleic Acids Res, 49:D751-D763, 2021, https://doi.org/10.1093/nar/gkaa939) comparative analysis system and provided for download via the JGI data portal (https://genome.jgi.doe.gov/portal/). This workflow scales to run on thousands of metagenome samples per year, which can vary by the complexity of microbial communities and sequencing depth. Here, we describe the different tools, databases, and parameters used at different steps of the workflow to help with the interpretation of metagenome data available in IMG and to enable researchers to apply this workflow to their own data. We use 20 publicly available sediment metagenomes to illustrate the computing requirements for the different steps and highlight the typical results of data processing. The workflow modules for read filtering and metagenome assembly are available as a workflow description language (WDL) file (https://code.jgi.doe.gov/BFoster/jgi_meta_wdl). The workflow modules for annotation and binning are provided as a service to the user community at https://img.jgi.doe.gov/submit and require filling out the project and associated metadata descriptions in the Genomes OnLine Database (GOLD) (S. Mukherjee, D. Stamatis, J. Bertsch, G. Ovchinnikova, et al., Nucleic Acids Res, 49:D723-D733, 2021, https://doi.org/10.1093/nar/gkaa983).IMPORTANCE The DOE JGI Metagenome Workflow is designed for processing metagenomic data sets starting from Illumina fastq files. It performs data preprocessing, error correction, assembly, structural and functional annotation, and binning. The results of processing are provided in several standard formats, such as fasta and gff, and can be used for subsequent integration into the Integrated Microbial Genomes and Microbiomes (IMG/M) system where they can be compared to a comprehensive set of publicly available metagenomes. As of 30 July 2020, 7,155 JGI metagenomes have been processed by the DOE JGI Metagenome Workflow. Here, we present a metagenome workflow developed at the JGI that generates rich data in standard formats and has been optimized for downstream analyses ranging from assessment of the functional and taxonomic composition of microbial communities to genome-resolved metagenomics and the identification and characterization of novel taxa. This workflow is currently being used to analyze thousands of metagenomic data sets in a consistent and standardized manner.

Genomes OnLine Database (GOLD) v.8: overview and updates.

The Genomes OnLine Database (GOLD) (https://gold.jgi.doe.gov/) is a manually curated, daily updated collection of genome projects and their metadata accumulated from around the world. The current version of the database includes over 1.17 million entries organized broadly into Studies (45 770), Organisms (387 382) or Biosamples (101 207), Sequencing Projects (355 364) and Analysis Projects (283 481). These four levels contain over 600 metadata fields, which includes 76 controlled vocabulary (CV) tables containing 3873 terms. GOLD provides an interactive web user interface for browsing and searching by a wide range of project and metadata fields. Users can enter details about their own projects in GOLD, which acts as a gatekeeper to ensure that metadata is accurately documented before submitting sequence information to the Integrated Microbial Genomes (IMG) system for analysis. In order to maintain a reference dataset for use by members of the scientific community, GOLD also imports projects from public repositories such as GenBank and SRA. The current status of the database, along with recent updates and improvements are described in this manuscript.