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1. Shotgun metagenomics captures more microbial diversity than targeted 16S rRNA gene sequencing for field specimens and preserved museum specimens

   https://doi.org/10.1371/journal.pone.0291540  

   Madison, Joseph D; LaBumbard, Brandon C; Woodhams, Douglas C (September 2023, PLOS ONE)

   Kalendar, Ruslan (Ed.)

   The use of museum specimens for research in microbial evolutionary ecology remains an under-utilized investigative dimension with important potential. Despite this potential, there remain barriers in methodology and analysis to the wide-spread adoption of museum specimens for such studies. Here, we hypothesized that there would be significant differences in taxonomic prediction and related diversity among sample type (museum or fresh) and sequencing strategy (medium-depth shotgun metagenomic or 16S rRNA gene). We found dramatically higher predicted diversity from shotgun metagenomics when compared to 16S rRNA gene sequencing in museum and fresh samples, with this differential being larger in museum specimens. Broadly confirming these hypotheses, the highest diversity found in fresh samples was with shotgun sequencing using the Rep200 reference inclusive of viruses and microeukaryotes, followed by the WoL reference database. In museum-specimens, community diversity metrics also differed significantly between sequencing strategies, with the alpha-diversity ACE differential being significantly greater than the same comparisons made for fresh specimens. Beta diversity results were more variable, with significance dependent on reference databases used. Taken together, these findings demonstrate important differences in diversity results and prompt important considerations for future experiments and downstream analyses aiming to incorporate microbiome datasets from museum specimens. 

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2. OGUs enable effective, phylogeny-aware analysis of even shallow metagenome community structures

   https://doi.org/2021.04.04.438427  

   Qiyun Zhu; Shi Huang; Antonio Gonzalez; Imran McGrath; Daniel McDonald; Niina Haiminen; George Armstrong; Yoshiki Vázquez-Baeza; Julian Yu; Justin Kuczynski; et al (April 2021, bioRxiv)

   We introduce Operational Genomic Unit (OGU), a metagenome analysis strategy that directly exploits sequence alignment hits to individual reference genomes as the minimum unit for assessing the diversity of microbial communities and their relevance to environmental factors. This approach is independent from taxonomic classification, granting the possibility of maximal resolution of community composition, and organizes features into an accurate hierarchy using a phylogenomic tree. The outputs are suitable for contemporary analytical protocols for community ecology, differential abundance and supervised learning while supporting phylogenetic methods, such as UniFrac and phylofactorization, that are seldomly applied to shotgun metagenomics despite being prevalent in 16S rRNA gene amplicon studies. As demonstrated in one synthetic and two real-world case studies, the OGU method produces biologically meaningful patterns from microbiome datasets. Such patterns further remain detectable at very low metagenomic sequencing depths. Compared with taxonomic unit-based analyses implemented in currently adopted metagenomics tools, and the analysis of 16S rRNA gene amplicon sequence variants, this method shows superiority in informing biologically relevant insights, including stronger correlation with body environment and host sex on the Human Microbiome Project dataset, and more accurate prediction of human age by the gut microbiomes in the Finnish population. We provide Woltka, a bioinformatics tool to implement this method, with full integration with the QIIME 2 package and the Qiita web platform, to facilitate OGU adoption in future metagenomics studies. Importance Shotgun metagenomics is a powerful, yet computationally challenging, technique compared to 16S rRNA gene amplicon sequencing for decoding the composition and structure of microbial communities. However, current analyses of metagenomic data are primarily based on taxonomic classification, which is limited in feature resolution compared to 16S rRNA amplicon sequence variant analysis. To solve these challenges, we introduce Operational Genomic Units (OGUs), which are the individual reference genomes derived from sequence alignment results, without further assigning them taxonomy. The OGU method advances current read-based metagenomics in two dimensions: (i) providing maximal resolution of community composition while (ii) permitting use of phylogeny-aware tools. Our analysis of real-world datasets shows several advantages over currently adopted metagenomic analysis methods and the finest-grained 16S rRNA analysis methods in predicting biological traits. We thus propose the adoption of OGU as standard practice in metagenomic studies. 

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3. Dissecting the dominant hot spring microbial populations based on community-wide sampling at single-cell genomic resolution

   https://doi.org/10.1038/s41396-021-01178-4  

   Bowers, Robert M.; Nayfach, Stephen; Schulz, Frederik; Jungbluth, Sean P.; Ruhl, Ilona A.; Sheremet, Andriy; Lee, Janey; Goudeau, Danielle; Eloe-Fadrosh, Emiley A.; Stepanauskas, Ramunas; et al (May 2022, The ISME Journal)

   Abstract With advances in DNA sequencing and miniaturized molecular biology workflows, rapid and affordable sequencing of single-cell genomes has become a reality. Compared to 16S rRNA gene surveys and shotgun metagenomics, large-scale application of single-cell genomics to whole microbial communities provides an integrated snapshot of community composition and function, directly links mobile elements to their hosts, and enables analysis of population heterogeneity of the dominant community members. To that end, we sequenced nearly 500 single-cell genomes from a low diversity hot spring sediment sample from Dewar Creek, British Columbia, and compared this approach to 16S rRNA gene amplicon and shotgun metagenomics applied to the same sample. We found that the broad taxonomic profiles were similar across the three sequencing approaches, though several lineages were missing from the 16S rRNA gene amplicon dataset, likely the result of primer mismatches. At the functional level, we detected a large array of mobile genetic elements present in the single-cell genomes but absent from the corresponding same species metagenome-assembled genomes. Moreover, we performed a single-cell population genomic analysis of the three most abundant community members, revealing differences in population structure based on mutation and recombination profiles. While the average pairwise nucleotide identities were similar across the dominant species-level lineages, we observed differences in the extent of recombination between these dominant populations. Most intriguingly, the creek’s Hydrogenobacter sp . population appeared to be so recombinogenic that it more closely resembled a sexual species than a clonally evolving microbe. Together, this work demonstrates that a randomized single-cell approach can be useful for the exploration of previously uncultivated microbes from community composition to population structure. 

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4. Microbial Community Profiling Protocol with Full‐length 16S rRNA Sequences and Emu

   https://doi.org/10.1002/cpz1.978  

   Curry, Kristen D.; Soriano, Sirena; Nute, Michael G.; Villapol, Sonia; Dilthey, Alexander; Treangen, Todd J. (March 2024, Current Protocols)

   Abstract 16S rRNA targeted amplicon sequencing is an established standard for elucidating microbial community composition. While high‐throughput short‐read sequencing can elicit only a portion of the 16S rRNA gene due to their limited read length, third generation sequencing can read the 16S rRNA gene in its entirety and thus provide more precise taxonomic classification. Here, we present a protocol for generating full‐length 16S rRNA sequences with Oxford Nanopore Technologies (ONT) and a microbial community profile with Emu. We select Emu for analyzing ONT sequences as it leverages information from the entire community to overcome errors due to incomplete reference databases and hardware limitations to ultimately obtain species‐level resolution. This pipeline provides a low‐cost solution for characterizing microbiome composition by exploiting real‐time, long‐read ONT sequencing and tailored software for accurate characterization of microbial communities. © 2024 Wiley Periodicals LLC. Basic Protocol: Microbial community profiling with Emu Support Protocol 1: Full‐length 16S rRNA microbial sequences with Oxford Nanopore Technologies sequencing platform Support Protocol 2: Building a custom reference database for Emu 

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5. Sea cucumber grazing linked to enrichment of anaerobic microbial metabolisms in coral reef sediments

   https://doi.org/10.1093/ismejo/wraf088  

   Maritan, Andrew J; Clements, Cody S; Pratte, Zoe A; Hay, Mark E; Stewart, Frank J (May 2025, The ISME Journal)

   Abstract Sea cucumbers have been overharvested world-wide, making assessments of their ecological effects challenging, but recent research demonstrated that sea cucumbers increase coral survival via disease suppression and were therefore important for facilitating reef health. The mechanisms underpinning the sea cucumber-coral interaction therefore are not well understood but are likely mediated through sea cucumber grazing of microbes from reef sediments. We explored how sea cucumber grazing alters the sediment microbiome by leveraging a healthy sea cucumber population on a reef in French Polynesia. We used quantitative PCR, 16S rRNA gene sequencing, and shotgun metagenomics to compare the sediment microbiome in cages placed in situ with or without sea cucumbers. We hypothesized that grazing would lower microbial biomass, change sediment microbiome composition, and deplete sediment metagenomes of anaerobic metabolisms, likely due to aeration of the sediments. Sea cucumber grazing resulted in a 75% reduction in 16S rRNA gene abundances and reshaped microbiome composition, causing a significant decrease of cyanobacteria and other phototrophs relative to ungrazed sediments. Grazing also resulted in a depletion of genes associated with cyanotoxin synthesis, suggesting a potential link to coral health. In contrast to expectations, grazed sediment metagenomes were enriched with marker genes of diverse anaerobic or microaerophilic metabolisms, including those encoding high oxygen affinity cytochrome oxidases. This enrichment differs from patterns linked to other bioturbating invertebrates. We hypothesize that grazing enriches anaerobic processes in sediment microbiomes through removal of oxygen-producing autotrophs, fecal deposition of sea cucumber gut-associated anaerobes, or modification of sediment diffusibility. These results suggest that sea cucumber harvesting influences biogeochemical processes in reef sediments, potentially mediating coral survival by altering the sediment microbiome and its production of coral-influencing metabolites. 

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