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1. Evaluating de Novo Assembly and Binning Strategies for Time Series Drinking Water Metagenomes

   https://doi.org/10.1128/Spectrum.01434-21  

   Vosloo, Solize ; Huo, Linxuan ; Anderson, Christopher L. ; Dai, Zihan ; Sevillano, Maria ; Pinto, Ameet ( December 2021 , Microbiology Spectrum)

   Gralnick, Jeffrey A. (Ed.)

   ABSTRACT Reconstructing microbial genomes from metagenomic short-read data can be challenging due to the unknown and uneven complexity of microbial communities. This complexity encompasses highly diverse populations, which often includes strain variants. Reconstructing high-quality genomes is a crucial part of the metagenomic workflow, as subsequent ecological and metabolic inferences depend on their accuracy, quality, and completeness. In contrast to microbial communities in other ecosystems, there has been no systematic assessment of genome-centric metagenomic workflows for drinking water microbiomes. In this study, we assessed the performance of a combination of assembly and binning strategies for time series drinking water metagenomes that were collected over 6 months. The goal of this study was to identify the combination of assembly and binning approaches that result in high-quality and -quantity metagenome-assembled genomes (MAGs), representing most of the sequenced metagenome. Our findings suggest that the metaSPAdes coassembly strategies had the best performance, as they resulted in larger and less fragmented assemblies, with at least 85% of the sequence data mapping to contigs greater than 1 kbp. Furthermore, a combination of metaSPAdes coassembly strategies and MetaBAT2 produced the highest number of medium-quality MAGs while capturing at least 70% of the metagenomes based on read recruitment. Utilizing different assembly/binningmore »approaches also assists in the reconstruction of unique MAGs from closely related species that would have otherwise collapsed into a single MAG using a single workflow. Overall, our study suggests that leveraging multiple binning approaches with different metaSPAdes coassembly strategies may be required to maximize the recovery of good-quality MAGs. IMPORTANCE Drinking water contains phylogenetic diverse groups of bacteria, archaea, and eukarya that affect the esthetic quality of water, water infrastructure, and public health. Taxonomic, metabolic, and ecological inferences of the drinking water microbiome depend on the accuracy, quality, and completeness of genomes that are reconstructed through the application of genome-resolved metagenomics. Using time series metagenomic data, we present reproducible genome-centric metagenomic workflows that result in high-quality and -quantity genomes, which more accurately signifies the sequenced drinking water microbiome. These genome-centric metagenomic workflows will allow for improved taxonomic and functional potential analysis that offers enhanced insights into the stability and dynamics of drinking water microbial communities.« less
2. Depth-discrete metagenomics reveals the roles of microbes in biogeochemical cycling in the tropical freshwater Lake Tanganyika

   https://doi.org/10.1038/s41396-021-00898-x  

   Tran, Patricia Q. ; Bachand, Samantha C. ; McIntyre, Peter B. ; Kraemer, Benjamin M. ; Vadeboncoeur, Yvonne ; Kimirei, Ismael A. ; Tamatamah, Rashid ; McMahon, Katherine D. ; Anantharaman, Karthik ( February 2021 , The ISME Journal)

   Lake Tanganyika (LT) is the largest tropical freshwater lake, and the largest body of anoxic freshwater on Earth’s surface. LT’s mixed oxygenated surface waters float atop a permanently anoxic layer and host rich animal biodiversity. However, little is known about microorganisms inhabiting LT’s 1470 meter deep water column and their contributions to nutrient cycling, which affect ecosystem-level function and productivity. Here, we applied genome-resolved metagenomics and environmental analyses to link specific taxa to key biogeochemical processes across a vertical depth gradient in LT. We reconstructed 523 unique metagenome-assembled genomes (MAGs) from 34 bacterial and archaeal phyla, including many rarely observed in freshwater lakes. We identified sharp contrasts in community composition and metabolic potential with an abundance of typical freshwater taxa in oxygenated mixed upper layers, and Archaea and uncultured Candidate Phyla in deep anoxic waters. Genomic capacity for nitrogen and sulfur cycling was abundant in MAGs recovered from anoxic waters, highlighting microbial contributions to the productive surface layers via recycling of upwelled nutrients, and greenhouse gases such as nitrous oxide. Overall, our study provides a blueprint for incorporation of aquatic microbial genomics in the representation of tropical freshwater lakes, especially in the context of ongoing climate change, which is predicted tomore »bring increased stratification and anoxia to freshwater lakes.

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3. Microbial colonization and persistence in deep fractured shales is guided by metabolic exchanges and viral predation

   https://doi.org/10.1186/s40168-021-01194-8  

   Amundson, Kaela K. ; Borton, Mikayla A. ; Daly, Rebecca A. ; Hoyt, David W. ; Wong, Allison ; Eder, Elizabeth ; Moore, Joseph ; Wunch, Kenneth ; Wrighton, Kelly C. ; Wilkins, Michael J. ( December 2022 , Microbiome)

   Abstract Background Microbial colonization of subsurface shales following hydraulic fracturing offers the opportunity to study coupled biotic and abiotic factors that impact microbial persistence in engineered deep subsurface ecosystems. Shale formations underly much of the continental USA and display geographically distinct gradients in temperature and salinity. Complementing studies performed in eastern USA shales that contain brine-like fluids, here we coupled metagenomic and metabolomic approaches to develop the first genome-level insights into ecosystem colonization and microbial community interactions in a lower-salinity, but high-temperature western USA shale formation. Results We collected materials used during the hydraulic fracturing process (i.e., chemicals, drill muds) paired with temporal sampling of water produced from three different hydraulically fractured wells in the STACK ( S ooner T rend A nadarko Basin, C anadian and K ingfisher) shale play in OK, USA. Relative to other shale formations, our metagenomic and metabolomic analyses revealed an expanded taxonomic and metabolic diversity of microorganisms that colonize and persist in fractured shales. Importantly, temporal sampling across all three hydraulic fracturing wells traced the degradation of complex polymers from the hydraulic fracturing process to the production and consumption of organic acids that support sulfate- and thiosulfate-reducing bacteria. Furthermore, we identified 5587 viral genomesmore »and linked many of these to the dominant, colonizing microorganisms, demonstrating the key role that viral predation plays in community dynamics within this closed, engineered system. Lastly, top-side audit sampling of different source materials enabled genome-resolved source tracking, revealing the likely sources of many key colonizing and persisting taxa in these ecosystems. Conclusions These findings highlight the importance of resource utilization and resistance to viral predation as key traits that enable specific microbial taxa to persist across fractured shale ecosystems. We also demonstrate the importance of materials used in the hydraulic fracturing process as both a source of persisting shale microorganisms and organic substrates that likely aid in sustaining the microbial community. Moreover, we showed that different physicochemical conditions (i.e., salinity, temperature) can influence the composition and functional potential of persisting microbial communities in shale ecosystems. Together, these results expand our knowledge of microbial life in deep subsurface shales and have important ramifications for management and treatment of microbial biomass in hydraulically fractured wells.« less
4. A consensus-based ensemble approach to improve transcriptome assembly

   https://doi.org/10.1186/s12859-021-04434-8  

   Voshall, Adam ; Behera, Sairam ; Li, Xiangjun ; Yu, Xiao-Hong ; Kapil, Kushagra ; Deogun, Jitender S. ; Shanklin, John ; Cahoon, Edgar B. ; Moriyama, Etsuko N. ( October 2021 , BMC Bioinformatics)

   Systems-level analyses, such as differential gene expression analysis, co-expression analysis, and metabolic pathway reconstruction, depend on the accuracy of the transcriptome. Multiple tools exist to perform transcriptome assembly from RNAseq data. However, assembling high quality transcriptomes is still not a trivial problem. This is especially the case for non-model organisms where adequate reference genomes are often not available. Different methods produce different transcriptome models and there is no easy way to determine which are more accurate. Furthermore, having alternative-splicing events exacerbates such difficult assembly problems. While benchmarking transcriptome assemblies is critical, this is also not trivial due to the general lack of true reference transcriptomes.

   In this study, we first provide a pipeline to generate a set of the simulated benchmark transcriptome and corresponding RNAseq data. Using the simulated benchmarking datasets, we compared the performance of various transcriptome assembly approaches including both de novo and genome-guided methods. The results showed that the assembly performance deteriorates significantly when alternative transcripts (isoforms) exist or for genome-guided methods when the reference is not available from the same genome. To improve the transcriptome assembly performance, leveraging the overlapping predictions between different assemblies, we present a new consensus-based ensemble transcriptome assembly approach, ConSemble.

   Without usingmore »a reference genome, ConSemble using four de novo assemblers achieved an accuracy up to twice as high as any de novo assemblers we compared. When a reference genome is available, ConSemble using four genome-guided assemblies removed many incorrectly assembled contigs with minimal impact on correctly assembled contigs, achieving higher precision and accuracy than individual genome-guided methods. Furthermore, ConSemble using de novo assemblers matched or exceeded the best performing genome-guided assemblers even when the transcriptomes included isoforms. We thus demonstrated that the ConSemble consensus strategy both for de novo and genome-guided assemblers can improve transcriptome assembly. The RNAseq simulation pipeline, the benchmark transcriptome datasets, and the script to perform the ConSemble assembly are all freely available from:http://bioinfolab.unl.edu/emlab/consemble/.

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5. Global patterns of diversity and metabolism of microbial communities in deep-sea hydrothermal vent deposits

   https://doi.org/10.1186/s40168-022-01424-7  

   Zhou, Zhichao ; St. John, Emily ; Anantharaman, Karthik ; Reysenbach, Anna-Louise ( December 2022 , Microbiome)

   When deep-sea hydrothermal fluids mix with cold oxygenated fluids, minerals precipitate out of solution and form hydrothermal deposits. These actively venting deep-sea hydrothermal deposits support a rich diversity of thermophilic microorganisms which are involved in a range of carbon, sulfur, nitrogen, and hydrogen metabolisms. Global patterns of thermophilic microbial diversity in deep-sea hydrothermal ecosystems have illustrated the strong connectivity between geological processes and microbial colonization, but little is known about the genomic diversity and physiological potential of these novel taxa. Here we explore this genomic diversity in 42 metagenomes from four deep-sea hydrothermal vent fields and a deep-sea volcano collected from 2004 to 2018 and document their potential implications in biogeochemical cycles.

   Our dataset represents 3635 metagenome-assembled genomes encompassing 511 novel and recently identified genera from deep-sea hydrothermal settings. Some of the novel bacterial (107) and archaeal genera (30) that were recently reported from the deep-sea Brothers volcano were also detected at the deep-sea hydrothermal vent fields, while 99 bacterial and 54 archaeal genera were endemic to the deep-sea Brothers volcano deposits. We report some of the first examples of medium- (≥ 50% complete, ≤ 10% contaminated) to high-quality (> 90% complete, < 5% contaminated) MAGs from phyla and families never previously identified, or poorlymore »sampled, from deep-sea hydrothermal environments. We greatly expand the novel diversity of Thermoproteia, Patescibacteria (Candidate Phyla Radiation, CPR), and Chloroflexota found at deep-sea hydrothermal vents and identify a small sampling of two potentially novel phyla, designated JALSQH01 and JALWCF01. Metabolic pathway analysis of metagenomes provides insights into the prevalent carbon, nitrogen, sulfur, and hydrogen metabolic processes across all sites and illustrates sulfur and nitrogen metabolic “handoffs” in community interactions. We confirm that Campylobacteria and Gammaproteobacteria occupy similar ecological guilds but their prevalence in a particular site is driven by shifts in the geochemical environment.

   Our study of globally distributed hydrothermal vent deposits provides a significant expansion of microbial genomic diversity associated with hydrothermal vent deposits and highlights the metabolic adaptation of taxonomic guilds. Collectively, our results illustrate the importance of comparative biodiversity studies in establishing patterns of shared phylogenetic diversity and physiological ecology, while providing many targets for enrichment and cultivation of novel and endemic taxa.

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