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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. Cloacal microbial diversity is associated with competitive phenotypes in socially polyandrous jacanas

   https://doi.org/10.1093/ornithology/ukaf031  

   Houtz, Jennifer L; Acosta, Kimberly A; Berlow, Mae; Lipshutz, Sara E (July 2025, Ornithology)

   The composition of host-associated microbial communities may correlate with the overall status of the host, including physiology and fitness. New bi-directional hypotheses suggest that sexual behaviors can shape, and be shaped by reproductive microbiomes, which may be particularly important for species with mating systems that feature strong sexual selection. These dynamics have been particularly understudied in female animals. Using 16S rRNA sequencing, we compared the cloacal microbiome of females and males from two socially polyandrous bird species that vary in the strength of sexual selection, Jacana spinosa (Northern Jacana) and J. jacana (Wattled Jacana). We hypothesized that the strength of sexual selection would shape cloacal microbial diversity, such that the more polyandrous J. spinosa would have a more diverse microbiome, and that microbiomes would be more diverse in females than in males. If the reproductive microbiome is indicative of competitive status, we also hypothesized that cloacal microbial diversity would be associated with competitive traits, including plasma testosterone levels, body mass, or weaponry. We found no differences in microbial alpha diversity between species or sexes, but we did find that microbial beta diversity significantly differed between species. We also found a positive relationship between microbial alpha diversity and testosterone in female J. spinosa. Future experiments are needed to explore the potential drivers of correlations between the cloacal microbiome and competitive phenotypes in socially polyandrous jacanas. 

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3. 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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4. 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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5. Orientia, Rickettsia, and the microbiome in rodent attached chiggers in North Carolina, USA

   https://doi.org/10.1371/journal.pone.0311698  

   Richardson, Elise A; Garshong, Reuben; Chen, Kaiying; Crossley, Dac; Mclean, Bryan S; Wasserberg, Gideon; Apperson, Charles S; Roe, R Michael; Ponnusamy, Loganathan (December 2024, PLOS ONE)

   Ganta, Roman R (Ed.)

   Chiggers are larval mites that pose a significant health risk globally via the spread of scrub typhus. However, fundamental studies into the bacterial microbiome in North America have never been considered. In this investigation, chiggers were collected in the wild from two locally common rodent host species (i.e.,Sigmodon hispidusandPeromyscus leucopus) in three different ecoregions of North Carolina (NC), United States to investigate the composition of their bacterial communities, including potential pathogens. DNA was extracted from the chiggers, and the V3-V4 regions of the bacterial 16S rRNA gene were sequenced using next-generation sequencing (NGS). Alpha diversity metrics revealed significant differences in bacterial diversity among different collection counties. Beta diversity metrics also revealed that bacterial communities across counties were significantly different, suggesting changes in the microbiome as the environment changed. Specifically, we saw that the two western NC collection counties had similar bacterial composition as did the two eastern collection counties. In addition, we found that the chigger microbiome bacterial diversity and composition differed between rodent host species. The 16S rRNA sequence reads were assigned to 64 phyla, 106 orders, 199 families, and 359 genera. The major bacterial phylum was Actinobacteria. The most abundant species were in the generaCorynebacterium,Propionibacterium, class ZB2, andMethylobacterium. Sequences derived from potential pathogens within the generaOrientiaandRickettsiawere also detected. Our findings provide the first insights into the ecology of chigger microbiomes in the US. Further research is required to determine if the potential pathogens found detected in chiggers are a threat to humans and wildlife. 

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