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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 bemore »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.« less
2. METABOLIC: high-throughput profiling of microbial genomes for functional traits, metabolism, biogeochemistry, and community-scale functional networks

   https://doi.org/10.1186/s40168-021-01213-8  

   Zhou, Zhichao ; Tran, Patricia Q. ; Breister, Adam M. ; Liu, Yang ; Kieft, Kristopher ; Cowley, Elise S. ; Karaoz, Ulas ; Anantharaman, Karthik ( February 2022 , Microbiome)

   Advances in microbiome science are being driven in large part due to our ability to study and infer microbial ecology from genomes reconstructed from mixed microbial communities using metagenomics and single-cell genomics. Such omics-based techniques allow us to read genomic blueprints of microorganisms, decipher their functional capacities and activities, and reconstruct their roles in biogeochemical processes. Currently available tools for analyses of genomic data can annotate and depict metabolic functions to some extent; however, no standardized approaches are currently available for the comprehensive characterization of metabolic predictions, metabolite exchanges, microbial interactions, and microbial contributions to biogeochemical cycling.

   We present METABOLIC (METabolic And BiogeOchemistry anaLyses In miCrobes), a scalable software to advance microbial ecology and biogeochemistry studies using genomes at the resolution of individual organisms and/or microbial communities. The genome-scale workflow includes annotation of microbial genomes, motif validation of biochemically validated conserved protein residues, metabolic pathway analyses, and calculation of contributions to individual biogeochemical transformations and cycles. The community-scale workflow supplements genome-scale analyses with determination of genome abundance in the microbiome, potential microbial metabolic handoffs and metabolite exchange, reconstruction of functional networks, and determination of microbial contributions to biogeochemical cycles. METABOLIC can take input genomes from isolates, metagenome-assembled genomes, ormore »single-cell genomes. Results are presented in the form of tables for metabolism and a variety of visualizations including biogeochemical cycling potential, representation of sequential metabolic transformations, community-scale microbial functional networks using a newly defined metric “MW-score” (metabolic weight score), and metabolic Sankey diagrams. METABOLIC takes ~ 3 h with 40 CPU threads to process ~ 100 genomes and corresponding metagenomic reads within which the most compute-demanding part of hmmsearch takes ~ 45 min, while it takes ~ 5 h to complete hmmsearch for ~ 3600 genomes. Tests of accuracy, robustness, and consistency suggest METABOLIC provides better performance compared to other software and online servers. To highlight the utility and versatility of METABOLIC, we demonstrate its capabilities on diverse metagenomic datasets from the marine subsurface, terrestrial subsurface, meadow soil, deep sea, freshwater lakes, wastewater, and the human gut.

   METABOLIC enables the consistent and reproducible study of microbial community ecology and biogeochemistry using a foundation of genome-informed microbial metabolism, and will advance the integration of uncultivated organisms into metabolic and biogeochemical models. METABOLIC is written in Perl and R and is freely available under GPLv3 athttps://github.com/AnantharamanLab/METABOLIC.

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3. Pseudomonas cultivated from Andropogon gerardii rhizosphere show functional potential for promoting plant host growth and drought resilience

   https://doi.org/10.1186/s12864-022-09019-0  

   Sarkar, Soumyadev ; Kamke, Abigail ; Ward, Kaitlyn ; Hartung, Eli ; Ran, Qinghong ; Feehan, Brandi ; Galliart, Matthew ; Jumpponen, Ari ; Johnson, Loretta ; Lee, Sonny T. M. ( November 2022 , BMC Genomics)

   Climate change will result in more frequent droughts that can impact soil-inhabiting microbiomes (rhizobiomes) in the agriculturally vital North American perennial grasslands. Rhizobiomes have contributed to enhancing drought resilience and stress resistance properties in plant hosts. In the predicted events of more future droughts, how the changing rhizobiome under environmental stress can impact the plant host resilience needs to be deciphered. There is also an urgent need to identify and recover candidate microorganisms along with their functions, involved in enhancing plant resilience, enabling the successful development of synthetic communities.

   In this study, we used the combination of cultivation and high-resolution genomic sequencing of bacterial communities recovered from the rhizosphere of a tallgrass prairie foundation grass,Andropogon gerardii. We cultivated the plant host-associated microbes under artificial drought-induced conditions and identified the microbe(s) that might play a significant role in the rhizobiome ofAndropogon gerardiiunder drought conditions. Phylogenetic analysis of the non-redundant metagenome-assembled genomes (MAGs) identified a bacterial genome of interest – MAG-Pseudomonas. Further metabolic pathway and pangenome analyses recovered genes and pathways related to stress responses including ACC deaminase; nitrogen transformation including assimilatory nitrate reductase in MAG-Pseudomonas,which might be associated with enhanced drought tolerance and growth forAndropogon gerardii.

   Our data indicated thatmore »the metagenome-assembled MAG-Pseudomonashas the functional potential to contribute to the plant host’s growth during stressful conditions. Our study also suggested the nitrogen transformation potential ofMAG-Pseudomonasthat could impactAndropogon gerardiigrowth in a positive way. The cultivation of MAG-Pseudomonassets the foundation to construct a successful synthetic community forAndropogon gerardii. To conclude, stress resilience mediated through genes ACC deaminase, nitrogen transformation potential through assimilatory nitrate reductase in MAG-Pseudomonascould place this microorganism as an important candidate of the rhizobiome aiding the plant host resilience under environmental stress. This study, therefore, provided insights into the MAG-Pseudomonasand its potential to optimize plant productivity under ever-changing climatic patterns, especially in frequent drought conditions.

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4. Optimizing de novo genome assembly from PCR-amplified metagenomes

   https://doi.org/10.7717/peerj.6902  

   Roux, Simon ; Trubl, Gareth ; Goudeau, Danielle ; Nath, Nandita ; Couradeau, Estelle ; Ahlgren, Nathan A. ; Zhan, Yuanchao ; Marsan, David ; Chen, Feng ; Fuhrman, Jed A. ; et al ( May 2019 , PeerJ)

   Metagenomics has transformed our understanding of microbial diversity across ecosystems, with recent advances enablingde novoassembly of genomes from metagenomes. These metagenome-assembled genomes are critical to provide ecological, evolutionary, and metabolic context for all the microbes and viruses yet to be cultivated. Metagenomes can now be generated from nanogram to subnanogram amounts of DNA. However, these libraries require several rounds of PCR amplification before sequencing, and recent data suggest these typically yield smaller and more fragmented assemblies than regular metagenomes.

   Here we evaluatede novoassembly methods of 169 PCR-amplified metagenomes, including 25 for which an unamplified counterpart is available, to optimize specific assembly approaches for PCR-amplified libraries. We first evaluated coverage bias by mapping reads from PCR-amplified metagenomes onto reference contigs obtained from unamplified metagenomes of the same samples. Then, we compared different assembly pipelines in terms of assembly size (number of bp in contigs ≥ 10 kb) and error rates to evaluate which are the best suited for PCR-amplified metagenomes.

   Read mapping analyses revealed that the depth of coverage within individual genomes is significantly more uneven in PCR-amplified datasets versus unamplified metagenomes, with regions of high depth of coverage enriched in short inserts. This enrichment scales with the number of PCRmore »cycles performed, and is presumably due to preferential amplification of short inserts. Standard assembly pipelines are confounded by this type of coverage unevenness, so we evaluated other assembly options to mitigate these issues. We found that a pipeline combining read deduplication and an assembly algorithm originally designed to recover genomes from libraries generated after whole genome amplification (single-cell SPAdes) frequently improved assembly of contigs ≥10 kb by 10 to 100-fold for low input metagenomes.

   PCR-amplified metagenomes have enabled scientists to explore communities traditionally challenging to describe, including some with extremely low biomass or from which DNA is particularly difficult to extract. Here we show that a modified assembly pipeline can lead to an improvedde novogenome assembly from PCR-amplified datasets, and enables a better genome recovery from low input metagenomes.

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5. Towards quantitative viromics for both double-stranded and single-stranded DNA viruses

   https://doi.org/10.7717/peerj.2777  

   Roux, Simon ; Solonenko, Natalie E. ; Dang, Vinh T. ; Poulos, Bonnie T. ; Schwenck, Sarah M. ; Goldsmith, Dawn B. ; Coleman, Maureen L. ; Breitbart, Mya ; Sullivan, Matthew B. ( December 2016 , PeerJ)

   Viruses strongly influence microbial population dynamics and ecosystem functions. However, our ability to quantitatively evaluate those viral impacts is limited to the few cultivated viruses and double-stranded DNA (dsDNA) viral genomes captured in quantitative viral metagenomes (viromes). This leaves the ecology of non-dsDNA viruses nearly unknown, including single-stranded DNA (ssDNA) viruses that have been frequently observed in viromes, but not quantified due to amplification biases in sequencing library preparations (Multiple Displacement Amplification, Linker Amplification or Tagmentation).

   Here we designed mock viral communities including both ssDNA and dsDNA viruses to evaluate the capability of a sequencing library preparation approach including an Adaptase step prior to Linker Amplification for quantitative amplification of both dsDNA and ssDNA templates. We then surveyed aquatic samples to provide first estimates of the abundance of ssDNA viruses.

   Mock community experiments confirmed the biased nature of existing library preparation methods for ssDNA templates (either largely enriched or selected against) and showed that the protocol using Adaptase plus Linker Amplification yielded viromes that were ±1.8-fold quantitative for ssDNA and dsDNA viruses. Application of this protocol to community virus DNA from three freshwater and three marine samples revealed that ssDNA viruses as a whole represent only a minor fraction (<5%)more »of DNA virus communities, though individual ssDNA genomes, both eukaryote-infecting Circular Rep-Encoding Single-Stranded DNA (CRESS-DNA) viruses and bacteriophages from theMicroviridaefamily, can be among the most abundant viral genomes in a sample.

   Together these findings provide empirical data for a new virome library preparation protocol, and a first estimate of ssDNA virus abundance in aquatic systems.

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