Flamholz ZN, Mulay SA, Leshyk V, Caporaso JG, Eisen JA, Kelly L,
The Microbial Dark Matter Symposium held on August 28-29, 2025, in Laguna Beach, Orange County, CA, convened a multidisciplinary group of scientists to address the vast unknowns in microbial life-from uncultured taxa and uncharacterized proteins to elusive viruses and spacefaring microbes. Set against a scenic coastal backdrop, the symposium highlighted advances in single-cell genomics, proximity ligation sequencing, and artificial intelligence-ready bioinformatics, while also probing the limits of microbial persistence, metabolism, and ecological distribution. Sessions explored microbial dark matter from multiple dimensions: cultivability, where new strategies are enabling recovery of elusive microbes; functional ambiguity, where metagenomic dark zones are illuminated by computational annotation; and genomic representation, where single-cell methods bridge gaps left by shotgun community sequencing. Researchers shared breakthroughs in identifying atmospheric microbiomes, "dark oxygen" production in groundwater ecosystems, and microbial survival on the International Space Station. The symposium emphasized integration of methods, disciplines, and ecosystems, advancing a collective push to illuminate the microbial dark matter on Earth and beyond.
This review introduces pharmacoexposomics as the environmental complement to pharmacogenomics — a framework that systematically quantifies the body's metabolic response to drug and environmental exposures using high-resolution mass spectrometry. It presents IndiPHARM, an initiative designed to integrate pharmacoexposomic data with pharmacogenomic data to advance precision medicine.
Byeongyeon Cho, Aleksandar D. Kostic, Braden T. Tierney, Chirag J. Patel
The oral microbiome interfaces humans and the environment and is implicated in diseases from caries to cardiovascular conditions. Yet, few studies systematically interrogate oral taxa associations with the host phenome and exposome in diverse populations. We developed a comprehensive oral microbiome atlas, deploying a Microbiome Association Study (MAS) evaluating relationships between host features including exposome, disease, and physiology and the microbiome in a 10,000-person representative US population. Evaluating demographics, 133 phenotypes, 473 exposures, and 20 disease outcomes across 1,349 taxa yielded >800k relationships and 45,757 FDR-significant associations. Age emerged as a major organizing axis, with genera following non-linear life-course patterns. Oral disease, smoking, and dietary sugar correlated with aciduric and anaerobic taxa, whereas oral health featured oxygen-tolerant Proteobacteria. The exposome and cardiovascular/respiratory disease linked to diverse taxa. These results establish the oral microbiome as a sensitive, population-scale indicator of the exposome, phenome, and systemic health.
Andrew G. Van Camp, Jiwoon Park, Elif Ozcelik, Onur Eskiocak, Kadir A. Ozler, Katie Papciak,
Dietary fat composition modulates host physiology and the gut microbiome, but the long-term effects of specific fat sources and the extent to which these changes resolve after dietary reversal remain incompletely defined. Here, we present a longitudinal multi-omic resource of mice maintained for one year on a purified control diet, seven high-fat diets differing in predominant fat source, or reversal regimens in which animals were switched from high-fat to control diet after 4 or 9 months. We further incorporated two cohorts with distinct pre-existing microbiome configurations to determine how baseline community structure shapes diet-induced remodeling of the gut microbiome ecosystem. By integrating longitudinal phenotyping, fecal metagenomics, fecal metabolomics, plasma metabolomics and lipidomics, and intestinal single-cell RNA sequencing, we defined the shared and dietary fat-specific responses across host and microbiome compartments. Baseline microbiome composition strongly influenced microbial responses to diet, indicating that pre-existing community structure is a major determinant of dietary ecosystem remodeling. Although many altered features shifted toward baseline after dietary reversal, only approximately half of diet-associated microbial changes recovered within the study window. A subset of taxa exhibited persistent alterations, including sustained depletion of Lactobacillus johnsonii and Bifidobacterium pseudolongum and sustained enrichment of Alistipes finegoldii, consistent with a "microbiome memory" of prior high-fat diet exposure. This memory effect is mirrored in the host, by sustained suppression of major histocompatibility complex class II (MHC-II) gene expression in intestinal epithelial cells after dietary reversal. These findings indicate that dietary fats leave a lasting imprint on the host-microbiome interactome that survives dietary intervention.
Kelliher JM, Mirzayi C, Bordenstein SR, Oliver A, Kellogg CA, Hatcher EL,
The interdisciplinary nature of microbiome research, coupled with the generation of complex multi-omics data, makes knowledge sharing challenging. The Strengthening the Organization and Reporting of Microbiome Studies (STORMS) guidelines provide a checklist for the reporting of study information, experimental design, and analytical methods within a scientific manuscript on human microbiome research. In this Consensus Statement, we present the Standards for Technical Reporting in Environmental and host-Associated Microbiome Studies (STREAMS) guidelines. The guidelines expand on STORMS and include 67 items to support the reporting and review of environmental (e.g., terrestrial, aquatic, atmospheric, engineered), synthetic, and non-human host-associated microbiome studies in a standardized and machine-actionable manner. Based on input from 248 researchers spanning 28 countries, we provide detailed guidance, including comparisons to STORMS, and case studies that demonstrate the usage of the STREAMS guidelines.
Krista A. Ryon, James R. Henriksen, April Johns, Sara C. Diana, Victor Boddy, Gaby E. Carpenter,
From medicines to materials, our planet's microbial diversity comprises an enormous wellspring of biotechnological potential. For centuries, microbiologists have developed tools for interrogating microbial function, ranging from microscopy and culturing to, more recently, metagenomics. However, deploying these tools during fieldwork requires substantial forward planning, interdisciplinary technical expertise, and plans for navigating permitting and the ethical implications of bioprospecting. To address these challenges, we built The Two Frontiers Project Handbook and OpenTools Resource, which aggregates our expertise in high-throughput sampling, sequencing, and culturing of microbes from thousands of samples. We provide our full suite of fieldwork methods as well as relevant software and hardware. We lay our standards for team roles and construction, general expedition planning, sample transport, permitting, and numerous other key aspects of executing a successful field campaign. The version-controlled resource is available at https://two-frontiers-project.github.io/ and is open for non-commercial use.
Gabriel A. Al-Ghalith, Krista A. Ryon, James R. Henriksen, David C. Danko, Brett Farthing, Matteo Marengo,
XTree is a k-mer-based aligner enabling rapid, memory-efficient alignment of sequencing reads to whole-genome reference databases with up to millions of genomes. Here, we detail XTree's performance on short and long read sequencing data and demonstrate its high accuracy across diverse bacterial, viral, and eukaryotic genomes. Benchmarking demonstrates superior and/or comparable precision and recall over existing tools, with more scalable indexing and efficient memory mapping. We additionally provide pre-indexed databases, including (1) the Genome Taxonomy Database (versions r214-r226), (2) representative GenBank fungi and protozoan genomes and (3) the Pan-Viral-Compendium, a bespoke data resource spanning 6.6 million, quality-controlled, viral genomes.
Gabriel A. Al-Ghalith, Krista A. Ryon, Erika Santoro, Adam Barno, Marco Casartelli, Helena Villela,
Metagenomic analysis of deeply sequenced, eukaryotic-dominant symbiotic communities can be difficult for many metagenomic workflows. Here, we present MAGUS, a bioinformatic toolkit that uses a suite of custom bioinformatic methods for iterative genome assembly and filtering of pan-domain communities, where eukaryotes, bacteria, viruses, and functionally annotated gene catalogs are resolved and analyzed over a series of interconnected, modular software components. We evaluated MAGUS using deeply sequenced (median depth: 579 million reads) ten samples of hard corals, soft corals, and hydrozoans, which comprise complex, eukaryote-dominated symbiotic communities. We successfully resolved phylogenetically comparable host (N = 10), algal (N = 6), bacterial (N = 55), and viral (N = 160,925) genomes, as well as a gene catalog comprising 15,369,684 non-redundant genes (7.6% functionally annotated). MAGUS is available on GitHub (https://github.com/two-frontiers-project/2FP_MAGUS/).
Michael M. Weinstein, Braden Tierney, Elaine Wolfe, Shuiquan Tang, Venu Lagishetty, Jonathan P. Jacobs,
Inconsistent cellular lysis is a major source of inaccuracy in microbial analysis; there is a need to develop objective and standardized evaluation of lysis efficiency across diverse methods. Using a mock microbial community as a ground truth, we built a bioinformatic and experimental toolkit for this purpose, comparing the performance of chemical, thermal, enzymatic, and various physical lysis protocols. We evaluated over 150 lysis conditions and observed the highest performance from mechanically- and materially-optimized bead-based approaches. To extend these findings to a practical setting, we demonstrated that the Firmicutes to Bacteroides ratio, a frequently used compositional metric in gut microbiome analysis, is biased heavily by insufficient lysis, indicating the importance of benchmarking ongoing methodological development.
Type 2 diabetes (T2D) progresses through heterogeneous pathways that glycemic staging alone does not resolve. We constructed the Metabolic Atlas of the Proteome in Diabetes (MAP-D), leveraging Olink measurements of 2,923 circulating proteins in ~42,000 UK Biobank participants to map associations with three cardiometabolic hallmarks — adiposity (BMI), a proxy for insulin resistance (triglyceride-to-HDL cholesterol ratio), and glycemia (HbA1c) — across normoglycemia, prediabetes, and incident T2D. We triangulated cross-sectional associations with bidirectional Mendelian randomization and semaglutide trial proteomics to infer causal directionality. This revealed three distinct causal architectures: adiposity predominantly reshapes the proteome, glycemia is driven by upstream proteins, and insulin resistance shows bidirectional feedback. Integration with trial data identified proteins reversed by therapy and a subset of persistent proteins that remain dysregulated despite GLP-1RA treatment. These persistent proteins are associated with incident coronary artery disease and overlap with targets of approved therapies, nominating candidates for combination strategies beyond GLP-1RA monotherapy.
Cyanobacteria are photosynthetic organisms that play important roles in carbon cycling and are promising bioproduction chassis. Here, we isolate two novel cyanobacteria with 4.6Mbp genomes, UTEX 3221 and UTEX 3222, from a unique marine environment with naturally elevated CO₂. We describe complete genome sequences for both isolates and, focusing on UTEX 3222 due to its planktonic growth in liquid, characterize biotechnologically relevant growth and biomass characteristics. UTEX 3222 outpaces other fast-growing model strains on a solid medium. It can double every 2.35 hours in a liquid medium and grows to high density (>31 g/L biomass dry weight) in batch culture, nearly double that of Synechococcus sp. PCC 11901, whose high-density growth was recently reported. In addition, UTEX 3222 sinks readily, settling more quickly than other fast-growing strains, suggesting favorable economics of harvesting UTEX 3222 biomass. These traits may make UTEX 3222 a compelling choice for marine carbon dioxide removal (CDR) and photosynthetic bioproduction from CO₂. Overall, we find that bio-prospecting in environments with naturally elevated CO₂ may uncover novel CO₂-metabolizing organisms with unique characteristics.
