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1. Evaluating and Improving Small Subunit rRNA PCR Primer Coverage for Bacteria, Archaea, and Eukaryotes Using Metagenomes from Global Ocean Surveys

   https://doi.org/10.1128/mSystems.00565-21  

   McNichol, Jesse; Berube, Paul M.; Biller, Steven J.; Fuhrman, Jed A. (June 2021, mSystems)

   Gilbert, Jack A. (Ed.)

   ABSTRACT Small subunit rRNA (SSU rRNA) amplicon sequencing can quantitatively and comprehensively profile natural microbiomes, representing a critically important tool for studying diverse global ecosystems. However, results will only be accurate if PCR primers perfectly match the rRNA of all organisms present. To evaluate how well marine microorganisms across all 3 domains are detected by this method, we compared commonly used primers with >300 million rRNA gene sequences retrieved from globally distributed marine metagenomes. The best-performing primers compared to 16S rRNA of bacteria and archaea were 515Y/926R and 515Y/806RB, which perfectly matched over 96% of all sequences. Considering cyanobacterial and chloroplast 16S rRNA, 515Y/926R had the highest coverage (99%), making this set ideal for quantifying marine primary producers. For eukaryotic 18S rRNA sequences, 515Y/926R also performed best (88%), followed by V4R/V4RB (18S rRNA specific; 82%)—demonstrating that the 515Y/926R combination performs best overall for all 3 domains. Using Atlantic and Pacific Ocean samples, we demonstrate high correspondence between 515Y/926R amplicon abundances (generated for this study) and metagenomic 16S rRNA (median R 2 = 0.98, n  = 272), indicating amplicons can produce equally accurate community composition data compared with shotgun metagenomics. Our analysis also revealed that expected performance of all primer sets could be improved with minor modifications, pointing toward a nearly completely universal primer set that could accurately quantify biogeochemically important taxa in ecosystems ranging from the deep sea to the surface. In addition, our reproducible bioinformatic workflow can guide microbiome researchers studying different ecosystems or human health to similarly improve existing primers and generate more accurate quantitative amplicon data. IMPORTANCE PCR amplification and sequencing of marker genes is a low-cost technique for monitoring prokaryotic and eukaryotic microbial communities across space and time but will work optimally only if environmental organisms match PCR primer sequences exactly. In this study, we evaluated how well primers match globally distributed short-read oceanic metagenomes. Our results demonstrate that primer sets vary widely in performance, and that at least for marine systems, rRNA amplicon data from some primers lack significant biases compared to metagenomes. We also show that it is theoretically possible to create a nearly universal primer set for diverse saline environments by defining a specific mixture of a few dozen oligonucleotides, and present a software pipeline that can guide rational design of primers for any environment with available meta’omic data. 

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2. TheOrganelle in theOintment: CrypticMitochondriaBiasCross-SpeciesMicrobiomeComparisons

   Dylan Sonett, Tanya Brown (May 2022, International Symposium on New Frontiers in Reef Coral Biotechnology (5 May 2022, Taiwan))

   The microbiomes of tropical corals are actively studied using 16S rRNA gene amplicons to understand microbial roles in coral health, metabolism, and disease resistance. However, due to the prokaryotic origins of mitochondria, primers targeting bacterial and archaeal 16S rRNA genes may also amplify homologous 12S mitochondrial rRNA genes from the host coral, associated microbial eukaryotes, and encrusting organisms. Standard microbial bioinformatics pipelines attempt to identify and remove these sequences by comparing them to reference taxonomies. However, commonly used tools have severely under-annotated mitochondrial sequences in 1440 coral microbiomes from the Global Coral Microbiome Project, preventing annotation of over 95% of reads in some samples. This issue persists when using Greengenes or SILVA prokaryotic reference taxonomies, and in other hosts, including 16S studies of vertebrates, and of marine sponges. Worse, mitochondrial under-annotation varies between coral families and across coral compartments, biasing comparisons of  - and  -diversity. By supplementing existing reference taxonomies with over 3000 animal mitochondrial rRNA gene sequences, we resolved roughly 97% of unique unclassified sequences as mitochondrial. These additional sequences did not cause a false elevation in mitochondrial annotations in mock communities with known compositions. We recommend using these extended taxonomies for coral microbiome analysis and whenever eukaryotic contamination may be a concern. 

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3. Contrasting diversity patterns of prokaryotes and protists over time and depth at the San-Pedro Ocean Time series

   https://doi.org/10.1038/s43705-022-00121-8  

   Yeh, Yi-Chun; Fuhrman, Jed A. (December 2022, ISME Communications)

   Abstract Community dynamics are central in microbial ecology, yet we lack studies comparing diversity patterns among marine protists and prokaryotes over depth and multiple years. Here, we characterized microbes at the San-Pedro Ocean Time series (2005–2018), using SSU rRNA gene sequencing from two size fractions (0.2–1 and 1–80 μm), with a universal primer set that amplifies from both prokaryotes and eukaryotes, allowing direct comparisons of diversity patterns in a single set of analyses. The 16S + 18S rRNA gene composition in the small size fraction was mostly prokaryotic (>92%) as expected, but the large size fraction unexpectedly contained 46–93% prokaryotic 16S rRNA genes. Prokaryotes and protists showed opposite vertical diversity patterns; prokaryotic diversity peaked at mid-depth, protistan diversity at the surface. Temporal beta-diversity patterns indicated prokaryote communities were much more stable than protists. Although the prokaryotic communities changed monthly, the average community stayed remarkably steady over 14 years, showing high resilience. Additionally, particle-associated prokaryotes were more diverse than smaller free-living ones, especially at deeper depths, contributed unexpectedly by abundant and diverse SAR11 clade II. Eukaryotic diversity was strongly correlated with the diversity of particle-associated prokaryotes but not free-living ones, reflecting that physical associations result in the strongest interactions, including symbioses, parasitism, and decomposer relationships. 

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4. Eukaryotic algal community composition in tropical environments from solar salterns to the open sea

   https://doi.org/10.3389/fmars.2023.1131351  

   Eckmann, Charlotte A.; Eberle, Jessica S.; Wittmers, Fabian; Wilken, Susanne; Bergauer, Kristin; Poirier, Camille; Blum, Marguerite; Makareviciute-Fichtner, Kriste; Jimenez, Valeria; Bachy, Charles; et al (June 2023, Frontiers in Marine Science)

   Tropical environments with unique abiotic and biotic factors—such as salt ponds, mangroves, and coral reefs—are often in close proximity. The heterogeneity of these environments is reflected in community shifts over short distances, resulting in high biodiversity. While phytoplankton assemblages physically associated with corals, particularly their symbionts, are well studied, less is known about phytoplankton diversity across tropical aquatic environments. We assess shifts in phytoplankton community composition along inshore to offshore gradients by sequencing and analyzing 16S rRNA gene amplicons using primers targeting the V1-V2 region that capture plastids from eukaryotic phytoplankton and cyanobacteria, as well as heterotrophic bacteria. Microbial alpha diversity computed from 16S V1-V2 amplicon sequence variant (ASV) data from 282 samples collected in and around Curaçao, in the Southern Caribbean Sea, varied more within the dynamic salt ponds, salterns, and mangroves, compared to the seemingly stable above-reef, off-reef, and open sea environments. Among eukaryotic phytoplankton, stramenopiles often exhibited the highest relative abundances in mangrove, above-reef, off-reef, and open sea environments, where cyanobacteria also showed high relative abundances. Within stramenopiles, diatom amplicons dominated in salt ponds and mangroves, while dictyochophytes and pelagophytes prevailed above reefs and offshore. Green algae and cryptophytes were also present, and the former exhibited transitions following the gradient from inland to offshore. Chlorophytes and prasinophyte Class IV dominated in salt ponds, while prasinophyte Class II, including Micromonas commoda and Ostreococcus Clade OII, had the highest relative abundances of green algae in mangroves, above-reef, off-reef, and the open sea. To improve Class II prasinophyte classification, we sequenced 18S rRNA gene amplicons from the V4 region in 41 samples which were used to interrelate plastid-based results with information on uncultured prasinophyte species from prior 18S rRNA gene-based studies. This highlighted the presence of newly described Ostreococcus bengalensis and two Micromonas candidate species. Network analyses identified co-occurrence patterns between individual phytoplankton groups, including cyanobacteria, and heterotrophic bacteria. Our study reveals multiple uncultured and novel lineages within green algae and dictyochophytes in tropical marine habitats. Collectively, the algal diversity patterns and potential co-occurrence relationships observed in connection to physicochemical and spatial influences help provide a baseline against which future change can be assessed. 

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5. Characterising Soil Eukaryotic Diversity From NEON Metagenomics Datasets

   https://doi.org/10.1111/1755-0998.70062  

   Vilonen, Leena; Thompson, Andrew; Adams, Byron; Ayres, Edward; Franco, André_L C; Wall, Diana H (January 2026, Molecular Ecology Resources)

   ABSTRACT Belowground eukaryotic diversity serves a vital role in soil ecosystem functioning, yet the composition, structure, and macroecology of these communities are significantly under‐characterized. The National Ecological Observatory Network (NEON) provides publicly available datasets from long‐term surveillance of numerous taxa and ecosystem properties. However, this dataset is not routinely evaluated for its eukaryotic component, likely because analyzing metagenomes for eukaryotic sequences is hampered by low relative sequence abundance, large genomes, poorer eukaryote representation in public reference databases, and is not yet mainstream. We mined the NEON soil metagenome datasets for 18S rRNA sequences using a custom‐built pipeline and produced a preliminary assessment of biodiversity trends in North American soil eukaryotes. We extracted ~800 18S rRNA reads per sample (~22,000 reads per site) from 1455 samples from 495 plots across 45 NEON sites in 11 biomes, which corresponded to 5183 genera in 35 phyla. To our knowledge, this represents the first large‐scale soil eukaryote analysis of NEON data. We asked whether taxonomic richness paralleled patterns previously established ecological trends and found that eukaryotic richness was negatively correlated with pH, managed sites lowered eukaryotic richness by 47%, most biomes had a distinct eukaryotic community, and fire decreased eukaryotic richness. These findings parallel generally accepted ecological trends and support the notion that NEON soil metagenome datasets can and should be used to explore spatiotemporal patterns in soil eukaryote diversity, its association with ecosystem functioning, and its response to environmental changes in North America. 

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