More Like this

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. 

   more » « less
2. 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. 

   more » « less
3. Microbiome Analysis Reveals Diversity and Function of Mollicutes Associated with the Eastern Oyster, Crassostrea virginica

   https://doi.org/10.1128/mSphere.00227-21  

   Pimentel, Zachary T.; Dufault-Thompson, Keith; Russo, Kayla T.; Scro, Abigail K.; Smolowitz, Roxanna M.; Gomez-Chiarri, Marta; Zhang, Ying (May 2021, mSphere)

   Campbell, Barbara J. (Ed.)

   Marine invertebrate microbiomes play important roles in diverse host and ecological processes. However, a mechanistic understanding of host-microbe interactions is currently available for a small number of model organisms. Here, an integrated taxonomic and functional analysis of the microbiome of the eastern oyster, Crassostrea virginica, was performed using 16S rRNA gene-based amplicon profiling, shotgun metagenomics, and genome-scale metabolic reconstruction. Relatively high variability of the microbiome was observed across individual oysters and among different tissue types. Specifically, a significantly higher alpha diversity was observed in the inner shell than in the gut, gill, mantle, and pallial fluid samples, and a distinct microbiome composition was revealed in the gut compared to other tissues examined in this study. Targeted metagenomic sequencing of the gut microbiota led to further characterization of a dominant bacterial taxon, the class Mollicutes, which was captured by the reconstruction of a metagenome-assembled genome (MAG). Genome-scale metabolic reconstruction of the oyster Mollicutes MAG revealed a reduced set of metabolic functions and a high reliance on the uptake of host-derived nutrients. A chitin degradation and an arginine deiminase pathway were unique to the MAG compared to closely related genomes of Mollicutes isolates, indicating distinct mechanisms of carbon and energy acquisition by the oyster-associated Mollicutes. A systematic reanalysis of public eastern oyster-derived microbiome data revealed a high prevalence of the Mollicutes among adult oyster guts and a significantly lower relative abundance of the Mollicutes in oyster larvae and adult oyster biodeposits. 

   more » « less
4. 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. 

   more » « less
5. 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 

   more » « less