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1. Analysis of metagenomic data

   https://doi.org/10.1038/s43586-024-00376-6  

   Liu, Shaopeng; Rodriguez, Judith S; Munteanu, Viorel; Ronkowski, Cynthia; Sharma, Nitesh Kumar; Alser, Mohammed; Andreace, Francesco; Blekhman, Ran; Błaszczyk, Dagmara; Chikhi, Rayan; et al (December 2025, Nature Reviews Methods Primers)

   Metagenomics has revolutionized our understanding of microbial communities, offering unprecedented insights into their genetic and functional diversity across Earth’s diverse ecosystems. Beyond their roles as environmental constituents, microbiomes act as symbionts, profoundly influencing the health and function of their host organisms. Given the inherent complexity of these communities and the diverse environments where they reside, the components of a metagenomics study must be carefully tailored to yield accurate results that are representative of the populations of interest. This Primer examines the methodological advancements and current practices that have shaped the field, from initial stages of sample collection and DNA extraction to the advanced bioinformatics tools employed for data analysis, with a particular focus on the profound impact of next-generation sequencing on the scale and accuracy of metagenomics studies. We critically assess the challenges and limitations inherent in metagenomics experimentation, available technologies and computational analysis methods. Beyond technical methodologies, we explore the application of metagenomics across various domains, including human health, agriculture and environmental monitoring. Looking ahead, we advocate for the development of more robust computational frameworks and enhanced interdisciplinary collaborations. This Primer serves as a comprehensive guide for advancing the precision and applicability of metagenomic studies, positioning them to address the complexities of microbial ecology and their broader implications for human health and environmental sustainability. 

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2. Metagenomics and the development of viral water quality tools

   https://doi.org/10.1038/s41545-019-0032-3  

   Bibby, Kyle; Crank, Katherine; Greaves, Justin; Li, Xiang; Wu, Zhenyu; Hamza, Ibrahim A.; Stachler, Elyse (March 2019, npj Clean Water)

   Abstract Human exposure to pathogenic viruses in environmental waters results in a significant global disease burden. Current microbial water quality monitoring approaches, mainly based on fecal indicator bacteria, insufficiently capture human health impacts posed by pathogenic viruses in water. The emergence of the ‘microbiome era’ and high-throughput metagenome sequencing has led to the discovery of novel human-associated viruses, including both pathogenic and commensal viruses in the human microbiome. The discovery of novel human-associated viruses is often followed by their detection in wastewater, highlighting the great diversity of human-associated viruses potentially present in the water environment. Novel human-associated viruses provide a rich reservoir to develop viral water quality management tools with diverse applications, such as regulating wastewater reuse and monitoring agricultural and recreational waters. Here, we review the pathway from viral discovery to water quality monitoring tool, and highlight select human-associated viruses identified by metagenomics and subsequently detected in the water environment (namely Bocavirus, Cosavirus, CrAssphage, Klassevirus, and Pepper Mild Mottle Virus). We also discuss research needs to enable the application of recently discovered human-associated viruses in water quality monitoring, including investigating the geographic distribution, environmental fate, and viability of potential indicator viruses. Examples suggest that recently discovered human pathogens are likely to be less abundant in sewage, while other human-associated viruses (e.g., bacteriophages or viruses from food) are more abundant but less human-specific. The improved resolution of human-associated viral diversity enabled by metagenomic tools provides a significant opportunity for improved viral water quality management tools. 

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3. Microbiome research: Application of “omics” technology

   https://doi.org/10.26717/BJSTR.2022.46.007300  

   Flowers, L. (September 2022, Biomedical journal of scientific technical research)

   Microbiome research is a thriving field focused on characterizing the composition and functionality of microbial populations or microbiomes from a wide array of ecological niches. Microbiomes occupy living organisms, soil, the atmosphere, and bodies of water and exist in moderate and extreme climates. Understanding the intractable microbial universes in various environments is challenging and potentially rewarding to humankind. Historically, elucidating pathogenic microbes and their impact on host species has dominated microbiome-based studies. Moreover, a tiny percentage of microbes can be cultured using classical culturing methods. With advancements in high throughput experimentation and computational tools derived from microbial ecology, there is a driving force to gain insight into the entire microbial consortium from various environmental and biological locations. Metagenomics, the study of all the microbial genomes in a sample using sequencing techniques (e.g., 16s rRNA amplicon sequencing and shotgun sequencing), has so far dominated the types of investigations conducted in the field of microbiome research. More recently, however, researchers are becoming increasingly interested in better understanding the complex microbe-associated molecular network and specific protein and metabolite functions associated with microbial genetic potential. Metaproteomic, meta transcriptomics, and metabolomics are three potent methods to accumulate information about microbial proteins, messenger RNA, and metabolites in a microbial community. These methods are currently being applied in laboratory settings to address our general lack of understanding of microbe-microbe interactions and microbe-environment interactions. 

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4. Microbial resolution of whole genome shotgun and 16S amplicon metagenomic sequencing using publicly available NEON data

   https://doi.org/doi.org/10.1371/journal.pone.0228899  

   Brumfield, Kyle D. (February 2020, PloS one)

   Microorganisms are ubiquitous in the biosphere, playing a crucial role in both biogeochemistry of the planet and human health. However, identifying these microorganisms and defining their function are challenging. Widely used approaches in comparative metagenomics, 16S amplicon sequencing and whole genome shotgun sequencing (WGS), have provided access to DNA sequencing analysis to identify microorganisms and evaluate diversity and abundance in various environments. However, advances in parallel high-throughput DNA sequencing in the past decade have introduced major hurdles, namely standardization of methods, data storage, reproducible interoperability of results, and data sharing. The National Ecological Observatory Network (NEON), established by the National Science Foundation, enables all researchers to address queries on a regional to continental scale around a variety of environmental challenges and provide high-quality, integrated, and standardized data from field sites across the U.S. As the amount of metagenomic data continues to grow, standardized procedures that allow results across projects to be assessed and compared is becoming increasingly important in the field of metagenomics. We demonstrate the feasibility of using publicly available NEON soil metagenomic sequencing datasets in combination with open access Metagenomics Rapid Annotation using the Subsystem Technology (MG-RAST) server to illustrate advantages of WGS compared to 16S amplicon sequencing. Four WGS and four 16S amplicon sequence datasets, from surface soil samples prepared by NEON investigators, were selected for comparison, using standardized protocols collected at the same locations in Colorado between April-July 2014. The dominant bacterial phyla detected across samples agreed between sequencing methodologies. However, WGS yielded greater microbial resolution, increased accuracy, and allowed identification of more genera of bacteria, archaea, viruses, and eukaryota, and putative functional genes that would have gone undetected using 16S amplicon sequencing. NEON open data will be useful for future studies characterizing and quantifying complex ecological processes associated with changing aquatic and terrestrial ecosystems. 

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5. Complementary Metagenomic Approaches Improve Reconstruction of Microbial Diversity in a Forest Soil

   https://doi.org/10.1128/mSystems.00768-19  

   Alteio, L. V.; Schulz, F.; Seshadri, R.; Varghese, N.; Rodriguez-Reillo, W.; Ryan, E.; Goudeau, D.; Eichorst, S. A.; Malmstrom, R. R.; Bowers, R. M.; et al (April 2020, mSystems)

   Jansson, Janet K. (Ed.)

   ABSTRACT Soil ecosystems harbor diverse microorganisms and yet remain only partially characterized as neither single-cell sequencing nor whole-community sequencing offers a complete picture of these complex communities. Thus, the genetic and metabolic potential of this “uncultivated majority” remains underexplored. To address these challenges, we applied a pooled-cell-sorting-based mini-metagenomics approach and compared the results to bulk metagenomics. Informatic binning of these data produced 200 mini-metagenome assembled genomes (sorted-MAGs) and 29 bulk metagenome assembled genomes (MAGs). The sorted and bulk MAGs increased the known phylogenetic diversity of soil taxa by 7.2% with respect to the Joint Genome Institute IMG/M database and showed clade-specific sequence recruitment patterns across diverse terrestrial soil metagenomes. Additionally, sorted-MAGs expanded the rare biosphere not captured through MAGs from bulk sequences, exemplified through phylogenetic and functional analyses of members of the phylum Bacteroidetes . Analysis of 67 Bacteroidetes sorted-MAGs showed conserved patterns of carbon metabolism across four clades. These results indicate that mini-metagenomics enables genome-resolved investigation of predicted metabolism and demonstrates the utility of combining metagenomics methods to tap into the diversity of heterogeneous microbial assemblages. IMPORTANCE Microbial ecologists have historically used cultivation-based approaches as well as amplicon sequencing and shotgun metagenomics to characterize microbial diversity in soil. However, challenges persist in the study of microbial diversity, including the recalcitrance of the majority of microorganisms to laboratory cultivation and limited sequence assembly from highly complex samples. The uncultivated majority thus remains a reservoir of untapped genetic diversity. To address some of the challenges associated with bulk metagenomics as well as low throughput of single-cell genomics, we applied flow cytometry-enabled mini-metagenomics to capture expanded microbial diversity from forest soil and compare it to soil bulk metagenomics. Our resulting data from this pooled-cell sorting approach combined with bulk metagenomics revealed increased phylogenetic diversity through novel soil taxa and rare biosphere members. In-depth analysis of genomes within the highly represented Bacteroidetes phylum provided insights into conserved and clade-specific patterns of carbon metabolism. 

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