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1
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Salmaso N, Cerasino L, Di Brizio M, Pindo M, Boscaini A. Phylogenomic and Pangenomic Assessment of a Mediterranean Strain of Raphidiopsis raciborskii Extends Knowledge of the Global Distribution and Characteristics of a Potentially Toxigenic Cyanobacterium. ENVIRONMENTAL MICROBIOLOGY REPORTS 2025; 17:e70098. [PMID: 40390271 PMCID: PMC12089652 DOI: 10.1111/1758-2229.70098] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/13/2025] [Revised: 04/08/2025] [Accepted: 04/16/2025] [Indexed: 05/21/2025]
Abstract
Among potentially toxigenic cyanobacteria, Raphidiopsis raciborskii has attracted considerable attention due to its ability to produce massive blooms and its recent spread to temperate regions. In this work, we reported for the first time a taxonomic and functional assessment of a R. raciborskii strain isolated from the Mediterranean region, contributing to filling a gap in the global distribution and characteristics of this species. The strain LT_0923 was isolated from Lake Trasimeno, a large and shallow lake in central Italy. The phylogenomic analyses based on selected marker genes and the core genome obtained from a pangenomic analysis based on a selection of available high-quality genomes showed a strong correspondence of the Lake Trasimeno strain with the North American and, at a lower average nucleotide identity, with the South American genomes. The LT_0923 genome did not show the presence of gene clusters encoding legacy cyanotoxins or emerging toxigenic compounds. The open pangenome and the large fraction of distinct gene families identified in the cloud and partly shell genome, enriched with genes specialised in environmental-specific functions and defence mechanisms, are consistent with the development of Raphidiopsis in geographically distinct regions.
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Affiliation(s)
- Nico Salmaso
- Research and Innovation CentreFondazione Edmund MachSan Michele all'AdigeItaly
- NBFCNational Biodiversity Future CenterPalermoItaly
| | - Leonardo Cerasino
- Research and Innovation CentreFondazione Edmund MachSan Michele all'AdigeItaly
| | | | - Massimo Pindo
- Research and Innovation CentreFondazione Edmund MachSan Michele all'AdigeItaly
| | - Adriano Boscaini
- Research and Innovation CentreFondazione Edmund MachSan Michele all'AdigeItaly
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2
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Shen Q, Chen Y, Zhao Y, Zhu Y, Xu C, Chang M, Gao Y, Ji F. Operational strategy for solid phase denitrification to achieve carbon balance between organic release and denitrification consumption. BIORESOURCE TECHNOLOGY 2025; 422:132239. [PMID: 39961518 DOI: 10.1016/j.biortech.2025.132239] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/19/2024] [Revised: 01/22/2025] [Accepted: 02/14/2025] [Indexed: 02/21/2025]
Abstract
This study developed a pilot-scale vertical-baffled solid-phase denitrification reactor (VbSPDR) incorporating polycaprolactone (PCL) and ceramsite as fillers to balance organic carbon release and denitrification consumption. The Box-Behnken design was employed to assess the effects of hydraulic retention time (HRT), temperature, and influent nitrate concentration on nitrate removal efficiency and COD accumulation. Optimal conditions yielded a 95 % nitrate removal rate at 33 °C, an HRT of 1.22 h, and an influent nitrate concentration of 19 mg/L. Conversely, effluent COD concentration was minimized and dropped 2.5 mg/L at 13 °C, an HRT of 0.39 h, and an influent nitrate concentration of 19 mg/L. The PCL layer enriched hydrolysis-acidification and heterotrophic denitrifying bacteria by unclassified_f__Comamonadaceae and Acidovorax, while heterotrophic genera Phreatobacter thrived in ceramsite layer, enhancing the metabolism of COD over-released from PCL. These findings indicate that inorganic fillers can effectively enhance nitrate removal and controll effluent COD under varied operational parameters.
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Affiliation(s)
- Qiushi Shen
- YANGTZE Eco-Environment Engineering Research Center, China Three Gorges Corporation, Wuhan 430012, PR China; National Engineering Research Center for Ecological Environment of Yangtze River Economic Belt, Wuhan 430012, PR China
| | - Yasong Chen
- YANGTZE Eco-Environment Engineering Research Center, China Three Gorges Corporation, Wuhan 430012, PR China; National Engineering Research Center for Ecological Environment of Yangtze River Economic Belt, Wuhan 430012, PR China
| | - Yunpeng Zhao
- YANGTZE Eco-Environment Engineering Research Center, China Three Gorges Corporation, Wuhan 430012, PR China; National Engineering Research Center for Ecological Environment of Yangtze River Economic Belt, Wuhan 430012, PR China
| | - Yating Zhu
- YANGTZE Eco-Environment Engineering Research Center, China Three Gorges Corporation, Wuhan 430012, PR China; National Engineering Research Center for Ecological Environment of Yangtze River Economic Belt, Wuhan 430012, PR China
| | - Chaowei Xu
- YANGTZE Eco-Environment Engineering Research Center, China Three Gorges Corporation, Wuhan 430012, PR China; National Engineering Research Center for Ecological Environment of Yangtze River Economic Belt, Wuhan 430012, PR China
| | - Manqi Chang
- YANGTZE Eco-Environment Engineering Research Center, China Three Gorges Corporation, Wuhan 430012, PR China; National Engineering Research Center for Ecological Environment of Yangtze River Economic Belt, Wuhan 430012, PR China
| | - Yanjin Gao
- YANGTZE Eco-Environment Engineering Research Center, China Three Gorges Corporation, Wuhan 430012, PR China; National Engineering Research Center for Ecological Environment of Yangtze River Economic Belt, Wuhan 430012, PR China
| | - Fangying Ji
- Key Laboratory of Three Gorges Reservoir Region's Eco-Environment, Ministry of Education, Chongqing University, Chongqing 400045, PR China.
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3
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Funnicelli MIG, de Carvalho LAL, Teheran-Sierra LG, Dibelli SC, Lemos EGDM, Pinheiro DG. Unveiling genomic features linked to traits of plant growth-promoting bacterial communities from sugarcane. THE SCIENCE OF THE TOTAL ENVIRONMENT 2024; 947:174577. [PMID: 38981540 DOI: 10.1016/j.scitotenv.2024.174577] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/09/2024] [Revised: 07/04/2024] [Accepted: 07/05/2024] [Indexed: 07/11/2024]
Abstract
Microorganisms are ubiquitous, and those inhabiting plants have been the subject of several studies. Plant-associated bacteria exhibit various biological mechanisms that enable them to colonize host plants and, in some cases, enhance their fitness. In this study, we describe the genomic features predicted to be associated with plant growth-promoting traits in six bacterial communities isolated from sugarcane. The use of highly accurate single-molecule real-time sequencing technology for metagenomic samples from these bacterial communities allowed us to recover 17 genomes. The taxonomic assignments for the binned genomes were performed, revealing taxa distributed across three main phyla: Bacillota, Bacteroidota, and Pseudomonadota, with the latter being the most representative. Subsequently, we functionally annotated the metagenome-assembled genomes (MAGs) to characterize their metabolic pathways related to plant growth-promoting traits. Our study successfully identified the enrichment of important functions related to phosphate and potassium acquisition, modulation of phytohormones, and mechanisms for coping with abiotic stress. These findings could be linked to the robust colonization of these sugarcane endophytes.
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Affiliation(s)
- Michelli Inácio Gonçalves Funnicelli
- Laboratory of Bioinformatics, Department of Agricultural, Livestock and Environmental Biotechnology, São Paulo State University (UNESP), School of Agricultural and Veterinary Sciences, Jaboticabal, SP, Brazil; Graduate Program in Agricultural and Livestock Microbiology, São Paulo State University (UNESP), School of Agricultural and Veterinary Sciences, Jaboticabal, SP, Brazil
| | - Lucas Amoroso Lopes de Carvalho
- Laboratory of Bioinformatics, Department of Agricultural, Livestock and Environmental Biotechnology, São Paulo State University (UNESP), School of Agricultural and Veterinary Sciences, Jaboticabal, SP, Brazil; Graduate Program in Agricultural and Livestock Microbiology, São Paulo State University (UNESP), School of Agricultural and Veterinary Sciences, Jaboticabal, SP, Brazil
| | - Luis Guillermo Teheran-Sierra
- Agronomy Research Program, Colombian Oil Palm Research Center, Cenipalma, Calle 98 No. 70-91, Piso 14, Bogotá 111121, Colombia
| | - Sabrina Custodio Dibelli
- Laboratory of Bioinformatics, Department of Agricultural, Livestock and Environmental Biotechnology, São Paulo State University (UNESP), School of Agricultural and Veterinary Sciences, Jaboticabal, SP, Brazil; Graduate Program in Agricultural and Livestock Microbiology, São Paulo State University (UNESP), School of Agricultural and Veterinary Sciences, Jaboticabal, SP, Brazil
| | - Eliana Gertrudes de Macedo Lemos
- Graduate Program in Agricultural and Livestock Microbiology, São Paulo State University (UNESP), School of Agricultural and Veterinary Sciences, Jaboticabal, SP, Brazil; Molecular Biology Laboratory, Institute for Research in Bioenergy (IPBEN), São Paulo State University (UNESP), School of Agricultural and Veterinary Sciences, Jaboticabal, SP, Brazil
| | - Daniel Guariz Pinheiro
- Laboratory of Bioinformatics, Department of Agricultural, Livestock and Environmental Biotechnology, São Paulo State University (UNESP), School of Agricultural and Veterinary Sciences, Jaboticabal, SP, Brazil; Graduate Program in Agricultural and Livestock Microbiology, São Paulo State University (UNESP), School of Agricultural and Veterinary Sciences, Jaboticabal, SP, Brazil.
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4
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Luan T, Cepeda V, Liu B, Bowen Z, Ayyangar U, Almeida M, Hill CM, Koren S, Treangen TJ, Porter A, Pop M. MetaCompass: Reference-guided Assembly of Metagenomes. ARXIV 2024:arXiv:2403.01578v1. [PMID: 38903742 PMCID: PMC11188144] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Subscribe] [Scholar Register] [Indexed: 06/22/2024]
Abstract
Metagenomic studies have primarily relied on de novo assembly for reconstructing genes and genomes from microbial mixtures. While reference-guided approaches have been employed in the assembly of single organisms, they have not been used in a metagenomic context. Here we describe the first effective approach for reference-guided metagenomic assembly that can complement and improve upon de novo metagenomic assembly methods for certain organisms. Such approaches will be increasingly useful as more genomes are sequenced and made publicly available.
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Affiliation(s)
- Tu Luan
- Department of Computer Science, University of Maryland, College Park, Maryland, USA
- Center for Bioinformatics and Computational Biology, University of Maryland, College Park, Maryland, USA
| | - Victoria Cepeda
- Department of Computer Science, University of Maryland, College Park, Maryland, USA
- Center for Bioinformatics and Computational Biology, University of Maryland, College Park, Maryland, USA
| | - Bo Liu
- Department of Computer Science, University of Maryland, College Park, Maryland, USA
- Center for Bioinformatics and Computational Biology, University of Maryland, College Park, Maryland, USA
| | - Zac Bowen
- Fraunhofer USA Center Mid-Atlantic, Riverdale, Maryland, USA
| | - Ujjwal Ayyangar
- Fraunhofer USA Center Mid-Atlantic, Riverdale, Maryland, USA
| | - Mathieu Almeida
- Center for Bioinformatics and Computational Biology, University of Maryland, College Park, Maryland, USA
| | - Christopher M. Hill
- Department of Computer Science, University of Maryland, College Park, Maryland, USA
- Center for Bioinformatics and Computational Biology, University of Maryland, College Park, Maryland, USA
| | - Sergey Koren
- Genome Informatics Section, Computational and Statistical Genomics Branch, National Human Genome Research Institute, Bethesda, Maryland, USA
| | - Todd J. Treangen
- Center for Bioinformatics and Computational Biology, University of Maryland, College Park, Maryland, USA
| | - Adam Porter
- Department of Computer Science, University of Maryland, College Park, Maryland, USA
| | - Mihai Pop
- Department of Computer Science, University of Maryland, College Park, Maryland, USA
- Center for Bioinformatics and Computational Biology, University of Maryland, College Park, Maryland, USA
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5
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Yancey CE, Mathiesen O, Dick GJ. Transcriptionally active nitrogen fixation and biosynthesis of diverse secondary metabolites by Dolichospermum and Aphanizomenon-like Cyanobacteria in western Lake Erie Microcystis blooms. HARMFUL ALGAE 2023; 124:102408. [PMID: 37164563 DOI: 10.1016/j.hal.2023.102408] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/04/2022] [Revised: 02/15/2023] [Accepted: 02/17/2023] [Indexed: 05/12/2023]
Abstract
Cyanobacterial harmful algal blooms (cyanoHABs) in the western basin of Lake Erie are dominated by microcystin producing Microcystis spp., but other cyanobacterial taxa that coexist in these communities may play important roles in production of toxins and shaping bloom dynamics and community function. In this study, we used metagenomic and metatranscriptomic data from the 2014 western Lake Erie cyanoHAB to explore the genetic diversity and biosynthetic potential of cyanobacteria belonging to the Anabaena, Dolichospermum, Aphanizomenon (ADA) clade. We reconstructed two near-complete metagenome-assembled genomes from two distinct ADA clade species, each containing biosynthetic gene clusters that encode novel and known secondary metabolites, including those with toxic and/or known taste and odor properties, that were transcriptionally active. However, neither ADA metagenome-assembled genome contained genes encoding guanitoxins, anatoxins, or saxitoxins, which are known to be produced by ADA. The ADA cyanobacteria accounted for most of the metagenomic and metatranscriptomic reads from nitrogen fixation genes, suggesting they were the dominant N-fixers at the times and stations sampled. Despite their relatively low abundance, our results highlight the possibility that ADA taxa could influence the water quality and ecology of Microcystis blooms, although the extent of these impacts remains to be quantified.
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Affiliation(s)
- Colleen E Yancey
- Department of Earth and Environmental Sciences, University of Michigan, Ann Arbor, 48109, USA
| | - Olivia Mathiesen
- Department of Earth and Environmental Sciences, University of Michigan, Ann Arbor, 48109, USA
| | - Gregory J Dick
- Department of Earth and Environmental Sciences, University of Michigan, Ann Arbor, 48109, USA; Cooperative Institute for Great Lakes Research (CIGLR), University of Michigan, 4840 South State Road, Ann Arbor, MI 48108 USA.
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6
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Jin H, Quan K, He Q, Kwok LY, Ma T, Li Y, Zhao F, You L, Zhang H, Sun Z. A high-quality genome compendium of the human gut microbiome of Inner Mongolians. Nat Microbiol 2023; 8:150-161. [PMID: 36604505 DOI: 10.1038/s41564-022-01270-1] [Citation(s) in RCA: 35] [Impact Index Per Article: 17.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2021] [Accepted: 10/13/2022] [Indexed: 01/07/2023]
Abstract
Metagenome-based resources have revealed the diversity and function of the human gut microbiome, but further understanding is limited by insufficient genome quality and a lack of samples from typically understudied populations. Here we used hybrid long-read PromethION and short-read HiSeq sequencing to characterize the faecal microbiota of 60 Inner Mongolian individuals (n = 180 samples over three time points) who were part of a probiotic yogurt intervention trial. We present the Inner Mongolian Gut Genome catalogue, comprising 802 closed and 5,927 high-quality metagenome-assembled genomes. This approach achieved high genome continuity and substantially increased the resolution of genomic elements, including ribosomal RNA operons, metabolic gene clusters, prophages and insertion sequences. Particularly, we report the ribosomal RNA operon copy numbers for uncultured species, over 12,000 previously undescribed gut prophages and the distribution of insertion sequence elements across gut bacteria. Overall, these data provide a high-quality, large-scale resource for studying the human gut microbiota.
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Affiliation(s)
- Hao Jin
- Inner Mongolia Key Laboratory of Dairy Biotechnology and Engineering, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China.,Key Laboratory of Dairy Products Processing, Ministry of Agriculture and Rural Affairs, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China.,Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China
| | - Keyu Quan
- Inner Mongolia Key Laboratory of Dairy Biotechnology and Engineering, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China.,Key Laboratory of Dairy Products Processing, Ministry of Agriculture and Rural Affairs, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China.,Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China
| | - Qiuwen He
- Inner Mongolia Key Laboratory of Dairy Biotechnology and Engineering, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China.,Key Laboratory of Dairy Products Processing, Ministry of Agriculture and Rural Affairs, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China.,Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China
| | - Lai-Yu Kwok
- Inner Mongolia Key Laboratory of Dairy Biotechnology and Engineering, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China.,Key Laboratory of Dairy Products Processing, Ministry of Agriculture and Rural Affairs, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China.,Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China
| | - Teng Ma
- Inner Mongolia Key Laboratory of Dairy Biotechnology and Engineering, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China.,Key Laboratory of Dairy Products Processing, Ministry of Agriculture and Rural Affairs, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China.,Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China
| | - Yalin Li
- Inner Mongolia Key Laboratory of Dairy Biotechnology and Engineering, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China.,Key Laboratory of Dairy Products Processing, Ministry of Agriculture and Rural Affairs, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China.,Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China
| | - Feiyan Zhao
- Inner Mongolia Key Laboratory of Dairy Biotechnology and Engineering, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China.,Key Laboratory of Dairy Products Processing, Ministry of Agriculture and Rural Affairs, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China.,Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China
| | - Lijun You
- Inner Mongolia Key Laboratory of Dairy Biotechnology and Engineering, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China.,Key Laboratory of Dairy Products Processing, Ministry of Agriculture and Rural Affairs, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China.,Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China
| | - Heping Zhang
- Inner Mongolia Key Laboratory of Dairy Biotechnology and Engineering, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China. .,Key Laboratory of Dairy Products Processing, Ministry of Agriculture and Rural Affairs, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China. .,Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China.
| | - Zhihong Sun
- Inner Mongolia Key Laboratory of Dairy Biotechnology and Engineering, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China. .,Key Laboratory of Dairy Products Processing, Ministry of Agriculture and Rural Affairs, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China. .,Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China.
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7
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Terrón-Camero LC, Gordillo-González F, Salas-Espejo E, Andrés-León E. Comparison of Metagenomics and Metatranscriptomics Tools: A Guide to Making the Right Choice. Genes (Basel) 2022; 13:2280. [PMID: 36553546 PMCID: PMC9777648 DOI: 10.3390/genes13122280] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2022] [Revised: 11/28/2022] [Accepted: 12/01/2022] [Indexed: 12/09/2022] Open
Abstract
The study of microorganisms is a field of great interest due to their environmental (e.g., soil contamination) and biomedical (e.g., parasitic diseases, autism) importance. The advent of revolutionary next-generation sequencing techniques, and their application to the hypervariable regions of the 16S, 18S or 23S ribosomal subunits, have allowed the research of a large variety of organisms more in-depth, including bacteria, archaea, eukaryotes and fungi. Additionally, together with the development of analysis software, the creation of specific databases (e.g., SILVA or RDP) has boosted the enormous growth of these studies. As the cost of sequencing per sample has continuously decreased, new protocols have also emerged, such as shotgun sequencing, which allows the profiling of all taxonomic domains in a sample. The sequencing of hypervariable regions and shotgun sequencing are technologies that enable the taxonomic classification of microorganisms from the DNA present in microbial communities. However, they are not capable of measuring what is actively expressed. Conversely, we advocate that metatranscriptomics is a "new" technology that makes the identification of the mRNAs of a microbial community possible, quantifying gene expression levels and active biological pathways. Furthermore, it can be also used to characterise symbiotic interactions between the host and its microbiome. In this manuscript, we examine the three technologies above, and discuss the implementation of different software and databases, which greatly impact the obtaining of reliable results. Finally, we have developed two easy-to-use pipelines leveraging Nextflow technology. These aim to provide everything required for an average user to perform a metagenomic analysis of marker genes with QIMME2 and a metatranscriptomic study using Kraken2/Bracken.
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Affiliation(s)
- Laura C. Terrón-Camero
- Bioinformatics Unit, Institute of Parasitology and Biomedicine “López-Neyra”, CSIC (IPBLN-CSIC), 18016 Granada, Spain
| | - Fernando Gordillo-González
- Bioinformatics Unit, Institute of Parasitology and Biomedicine “López-Neyra”, CSIC (IPBLN-CSIC), 18016 Granada, Spain
| | - Eduardo Salas-Espejo
- Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Granada, 18071 Granada, Spain
| | - Eduardo Andrés-León
- Bioinformatics Unit, Institute of Parasitology and Biomedicine “López-Neyra”, CSIC (IPBLN-CSIC), 18016 Granada, Spain
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8
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Obieze CC, Wani GA, Shah MA, Reshi ZA, Comeau AM, Khasa DP. Anthropogenic activities and geographic locations regulate microbial diversity, community assembly and species sorting in Canadian and Indian freshwater lakes. THE SCIENCE OF THE TOTAL ENVIRONMENT 2022; 826:154292. [PMID: 35248630 DOI: 10.1016/j.scitotenv.2022.154292] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/14/2021] [Revised: 02/13/2022] [Accepted: 02/28/2022] [Indexed: 06/14/2023]
Abstract
Freshwater lakes are important reservoirs and sources of drinking water globally. However, the microbiota, which supports the functionality of these ecosystems is threatened by the influx of nutrients, heavy metals and other toxic chemical substances from anthropogenic activities. The influence of these factors on the diversity, assembly mechanisms and co-occurrence patterns of bacterial communities in freshwater lakes is not clearly understood. Hence, samples were collected from six different impacted lakes in Canada and India and examined by 454-pyrosequencing technology. The trophic status of these lakes was determined using specific chemical parameters. Our results revealed that bacterial diversity and community composition was altered by both the lake water chemistry and geographic distance. Anthropogenic activities pervasively influenced species distribution. Dispersal limitation (32.3%), homogenous selection (31.8%) and drift (20%) accounted for the largest proportions of the bacterial community assembly mechanisms. Homogenous selection increased in lakes with higher nutrient concentration, while stochasticity reduced. Community functional profiles revealed that deterministic processes dominated the assembly mechanisms of phylotypes with higher potential for biodegradation, while stochasticity dominated the assembly of phylotypes with potential for antimicrobial resistance. Bacteroidota (44%) and Proteobacteria (34%) were the most abundant phyla. Co-occurrence network analysis revealed that complexity increased in more impacted lakes, while competition and the nature of anthropogenic activity contributed to species sorting. Overall, this study demonstrates that bacterial community changes in freshwater lakes are linked to anthropogenic activities, with corresponding consequences on the distribution of phylotypes of environmental and human health interest.
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Affiliation(s)
- Chinedu C Obieze
- Centre for Forest Research, Institute of Integrative Biology and Systems, Université Laval, Quebec, QC G1V0A6, Canada.
| | - Gowher A Wani
- Centre for Forest Research, Institute of Integrative Biology and Systems, Université Laval, Quebec, QC G1V0A6, Canada; Department of Botany, University of Kashmir, Srinagar 190006, Jammu and Kashmir, India
| | - Manzoor A Shah
- Department of Botany, University of Kashmir, Srinagar 190006, Jammu and Kashmir, India
| | - Zafar A Reshi
- Department of Botany, University of Kashmir, Srinagar 190006, Jammu and Kashmir, India
| | - André M Comeau
- Integrated Microbiome Resource, Dalhousie University, Halifax, NS, Canada
| | - Damase P Khasa
- Centre for Forest Research, Institute of Integrative Biology and Systems and Canada Research Chair in Forest Genomics, Université Laval, Quebec, QC G1V0A6, Canada
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9
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Ye L, Dong N, Xiong W, Li J, Li R, Heng H, Chan EWC, Chen S. High-Resolution Metagenomics of Human Gut Microbiota Generated by Nanopore and Illumina Hybrid Metagenome Assembly. Front Microbiol 2022; 13:801587. [PMID: 35633679 PMCID: PMC9134245 DOI: 10.3389/fmicb.2022.801587] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2021] [Accepted: 04/11/2022] [Indexed: 11/15/2022] Open
Abstract
Metagenome assembly is a core yet methodologically challenging step for taxonomic classification and functional annotation of a microbiome. This study aims to generate the high-resolution human gut metagenome using both Illumina and Nanopore platforms. Assembly was achieved using four assemblers, including Flye (Nanopore), metaSPAdes (Illumina), hybridSPAdes (Illumina and Nanopore), and OPERA-MS (Illumina and Nanopore). Hybrid metagenome assembly was shown to generate contigs with almost same sizes comparable to those produced using Illumina reads alone, but was more contiguous, informative, and longer compared with those assembled with Illumina reads only. In addition, hybrid metagenome assembly enables us to obtain complete plasmid sequences and much more AMR gene-encoding contigs than the Illumina method. Most importantly, using our workflow, 58 novel high-quality metagenome bins were obtained from four assembly algorithms, particularly hybrid assembly (47/58), although metaSPAdes could provide 11 high-quality bins independently. Among them, 29 bins were currently uncultured bacterial metagenome-assembled genomes. These findings were highly consistent and supported by mock community data tested. In the analysis of biosynthetic gene clusters (BGCs), the number of BGCs in the contigs from hybridSPAdes (241) is higher than that of contigs from metaSPAdes (233). In conclusion, hybrid metagenome assembly could significantly enhance the efficiency of contig assembly, taxonomic binning, and genome construction compared with procedures using Illumina short-read data alone, indicating that nanopore long reads are highly useful in metagenomic applications. This technique could be used to create high-resolution references for future human metagenome studies.
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Affiliation(s)
- Lianwei Ye
- Department of Infectious Diseases and Public Health, Jockey Club College of Veterinary Medicine and Life Sciences, City University of Hong Kong, Kowloon, Hong Kong SAR, China
| | - Ning Dong
- Department of Infectious Diseases and Public Health, Jockey Club College of Veterinary Medicine and Life Sciences, City University of Hong Kong, Kowloon, Hong Kong SAR, China
| | - Wenguang Xiong
- College of Veterinary Medicine, South China Agricultural University, Guangzhou, China
| | - Jun Li
- Department of Infectious Diseases and Public Health, Jockey Club College of Veterinary Medicine and Life Sciences, City University of Hong Kong, Kowloon, Hong Kong SAR, China
| | - Runsheng Li
- Department of Infectious Diseases and Public Health, Jockey Club College of Veterinary Medicine and Life Sciences, City University of Hong Kong, Kowloon, Hong Kong SAR, China
| | - Heng Heng
- Department of Infectious Diseases and Public Health, Jockey Club College of Veterinary Medicine and Life Sciences, City University of Hong Kong, Kowloon, Hong Kong SAR, China
| | - Edward Wai Chi Chan
- State Key Laboratory of Chemical Biology and Drug Discovery, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Hong Kong SAR, China
| | - Sheng Chen
- Department of Infectious Diseases and Public Health, Jockey Club College of Veterinary Medicine and Life Sciences, City University of Hong Kong, Kowloon, Hong Kong SAR, China
- Hong Kong Branch of the Southern Marine Science and Engineering Guangdong Laboratory, Guangzhou, China
- *Correspondence: Sheng Chen,
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10
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Liu S, Moon CD, Zheng N, Huws S, Zhao S, Wang J. Opportunities and challenges of using metagenomic data to bring uncultured microbes into cultivation. MICROBIOME 2022; 10:76. [PMID: 35546409 PMCID: PMC9097414 DOI: 10.1186/s40168-022-01272-5] [Citation(s) in RCA: 99] [Impact Index Per Article: 33.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/13/2022] [Accepted: 04/10/2022] [Indexed: 05/12/2023]
Abstract
Although there is now an extensive understanding of the diversity of microbial life on earth through culture-independent metagenomic DNA sequence analyses, the isolation and cultivation of microbes remains critical to directly study them and confirm their metabolic and physiological functions, and their ecological roles. The majority of environmental microbes are as yet uncultured however; therefore, bringing these rare or poorly characterized groups into culture is a priority to further understand microbiome functions. Moreover, cultivated isolates may find utility in a range of applications, such as new probiotics, biocontrol agents, and agents for industrial processes. The growing abundance of metagenomic and meta-transcriptomic sequence information from a wide range of environments provides more opportunities to guide the isolation and cultivation of microbes of interest. In this paper, we discuss a range of successful methodologies and applications that have underpinned recent metagenome-guided isolation and cultivation of microbe efforts. These approaches include determining specific culture conditions to enrich for taxa of interest, to more complex strategies that specifically target the capture of microbial species through antibody engineering and genome editing strategies. With the greater degree of genomic information now available from uncultivated members, such as via metagenome-assembled genomes, the theoretical understanding of their cultivation requirements will enable greater possibilities to capture these and ultimately gain a more comprehensive understanding of the microbiomes. Video Abstract.
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Affiliation(s)
- Sijia Liu
- State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, No. 2 Yuanmingyuan West Road, Haidian, Beijing, 100193, China
- College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou, 730020, China
| | - Christina D Moon
- AgResearch Ltd., Grasslands Research Centre, Palmerston North, New Zealand
| | - Nan Zheng
- State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, No. 2 Yuanmingyuan West Road, Haidian, Beijing, 100193, China
| | - Sharon Huws
- School of Biological Sciences and Institute for Global Food Security, 19 Chlorine Gardens, Queen's University Belfast, Belfast, UK
| | - Shengguo Zhao
- State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, No. 2 Yuanmingyuan West Road, Haidian, Beijing, 100193, China.
| | - Jiaqi Wang
- State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, No. 2 Yuanmingyuan West Road, Haidian, Beijing, 100193, China.
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11
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Adler A, Poirier S, Pagni M, Maillard J, Holliger C. Disentangle genus microdiversity within a complex microbial community by using a multi-distance long-read binning method: example of Candidatus Accumulibacter. Environ Microbiol 2022; 24:2136-2156. [PMID: 35315560 PMCID: PMC9311429 DOI: 10.1111/1462-2920.15947] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2021] [Accepted: 02/19/2022] [Indexed: 11/26/2022]
Abstract
Complete genomes can be recovered from metagenomes by assembling and binning DNA sequences into metagenome assembled genomes (MAGs). Yet, the presence of microdiversity can hamper the assembly and binning processes, possibly yielding chimeric, highly fragmented and incomplete genomes. Here, the metagenomes of four samples of aerobic granular sludge bioreactors containing Candidatus (Ca.) Accumulibacter, a phosphate-accumulating organism of interest for wastewater treatment, were sequenced with both PacBio and Illumina. Different strategies of genome assembly and binning were investigated, including published protocols and a binning procedure adapted to the binning of long contigs (MuLoBiSC). Multiple criteria were considered to select the best strategy for Ca. Accumulibacter, whose multiple strains in every sample represent a challenging microdiversity. In this case, the best strategy relies on long-read only assembly and a custom binning procedure including MuLoBiSC in metaWRAP. Several high-quality Ca. Accumulibacter MAGs, including a novel species, were obtained independently from different samples. Comparative genomic analysis showed that MAGs retrieved in different samples harbour genomic rearrangements in addition to accumulation of point mutations. The microdiversity of Ca. Accumulibacter, likely driven by mobile genetic elements, causes major difficulties in recovering MAGs, but it is also a hallmark of the panmictic lifestyle of these bacteria.
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Affiliation(s)
- Aline Adler
- Laboratory for Environmental Biotechnology, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
| | - Simon Poirier
- Laboratory for Environmental Biotechnology, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
| | - Marco Pagni
- Laboratory for Environmental Biotechnology, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.,Vital-IT Group, SIB Swiss Institute of Bioinformatics, Lausanne, Switzerland
| | - Julien Maillard
- Laboratory for Environmental Biotechnology, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.,IFP Energie nouvelles, 1 et 4 avenue de Bois-Préau, 92852, Rueil-Malmaison Cedex, France
| | - Christof Holliger
- Laboratory for Environmental Biotechnology, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
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12
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Peterson D, Bonham KS, Rowland S, Pattanayak CW, Klepac-Ceraj V. Comparative Analysis of 16S rRNA Gene and Metagenome Sequencing in Pediatric Gut Microbiomes. Front Microbiol 2021; 12:670336. [PMID: 34335499 PMCID: PMC8320171 DOI: 10.3389/fmicb.2021.670336] [Citation(s) in RCA: 66] [Impact Index Per Article: 16.5] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2021] [Accepted: 05/28/2021] [Indexed: 01/04/2023] Open
Abstract
The colonization of the human gut microbiome begins at birth, and over time, these microbial communities become increasingly complex. Most of what we currently know about the human microbiome, especially in early stages of development, was described using culture-independent sequencing methods that allow us to identify the taxonomic composition of microbial communities using genomic techniques, such as amplicon or shotgun metagenomic sequencing. Each method has distinct tradeoffs, but there has not been a direct comparison of the utility of these methods in stool samples from very young children, which have different features than those of adults. We compared the effects of profiling the human infant gut microbiome with 16S rRNA amplicon vs. shotgun metagenomic sequencing techniques in 338 fecal samples; younger than 15, 15-30, and older than 30 months of age. We demonstrate that observed changes in alpha-diversity and beta-diversity with age occur to similar extents using both profiling methods. We also show that 16S rRNA profiling identified a larger number of genera and we find several genera that are missed or underrepresented by each profiling method. We present the link between alpha diversity and shotgun metagenomic sequencing depth for children of different ages. These findings provide a guide for selecting an appropriate method and sequencing depth for the three studied age groups.
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Affiliation(s)
- Danielle Peterson
- Department of Biological Sciences, Wellesley College, Wellesley, MA, United States
| | - Kevin S Bonham
- Department of Biological Sciences, Wellesley College, Wellesley, MA, United States
| | - Sophie Rowland
- Department of Biological Sciences, Wellesley College, Wellesley, MA, United States
| | - Cassandra W Pattanayak
- Department of Mathematics, Quantitative Reasoning Program, and the Quantitative Analysis Institute at Wellesley College, Wellesley, MA, United States
| | | | - Vanja Klepac-Ceraj
- Department of Biological Sciences, Wellesley College, Wellesley, MA, United States
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13
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Garner E, Davis BC, Milligan E, Blair MF, Keenum I, Maile-Moskowitz A, Pan J, Gnegy M, Liguori K, Gupta S, Prussin AJ, Marr LC, Heath LS, Vikesland PJ, Zhang L, Pruden A. Next generation sequencing approaches to evaluate water and wastewater quality. WATER RESEARCH 2021; 194:116907. [PMID: 33610927 DOI: 10.1016/j.watres.2021.116907] [Citation(s) in RCA: 49] [Impact Index Per Article: 12.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/26/2020] [Revised: 01/15/2021] [Accepted: 02/03/2021] [Indexed: 05/24/2023]
Abstract
The emergence of next generation sequencing (NGS) is revolutionizing the potential to address complex microbiological challenges in the water industry. NGS technologies can provide holistic insight into microbial communities and their functional capacities in water and wastewater systems, thus eliminating the need to develop a new assay for each target organism or gene. However, several barriers have hampered wide-scale adoption of NGS by the water industry, including cost, need for specialized expertise and equipment, challenges with data analysis and interpretation, lack of standardized methods, and the rapid pace of development of new technologies. In this critical review, we provide an overview of the current state of the science of NGS technologies as they apply to water, wastewater, and recycled water. In addition, a systematic literature review was conducted in which we identified over 600 peer-reviewed journal articles on this topic and summarized their contributions to six key areas relevant to the water and wastewater fields: taxonomic classification and pathogen detection, functional and catabolic gene characterization, antimicrobial resistance (AMR) profiling, bacterial toxicity characterization, Cyanobacteria and harmful algal bloom identification, and virus characterization. For each application, we have presented key trends, noteworthy advancements, and proposed future directions. Finally, key needs to advance NGS technologies for broader application in water and wastewater fields are assessed.
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Affiliation(s)
- Emily Garner
- Wadsworth Department of Civil and Environmental Engineering, West Virginia University, 1306 Evansdale Drive, Morgantown, WV 26505, United States.
| | - Benjamin C Davis
- Charles E. Via, Jr. Department of Civil and Environmental Engineering, Virginia Tech, 1145 Perry Street, Blacksburg, VA 24061, United States
| | - Erin Milligan
- Charles E. Via, Jr. Department of Civil and Environmental Engineering, Virginia Tech, 1145 Perry Street, Blacksburg, VA 24061, United States
| | - Matthew Forrest Blair
- Charles E. Via, Jr. Department of Civil and Environmental Engineering, Virginia Tech, 1145 Perry Street, Blacksburg, VA 24061, United States
| | - Ishi Keenum
- Charles E. Via, Jr. Department of Civil and Environmental Engineering, Virginia Tech, 1145 Perry Street, Blacksburg, VA 24061, United States
| | - Ayella Maile-Moskowitz
- Charles E. Via, Jr. Department of Civil and Environmental Engineering, Virginia Tech, 1145 Perry Street, Blacksburg, VA 24061, United States
| | - Jin Pan
- Charles E. Via, Jr. Department of Civil and Environmental Engineering, Virginia Tech, 1145 Perry Street, Blacksburg, VA 24061, United States
| | - Mariah Gnegy
- Charles E. Via, Jr. Department of Civil and Environmental Engineering, Virginia Tech, 1145 Perry Street, Blacksburg, VA 24061, United States
| | - Krista Liguori
- Charles E. Via, Jr. Department of Civil and Environmental Engineering, Virginia Tech, 1145 Perry Street, Blacksburg, VA 24061, United States
| | - Suraj Gupta
- The Interdisciplinary PhD Program in Genetics, Bioinformatics, and Computational Biology, Virginia Tech, Blacksburg, VA 24061, United States
| | - Aaron J Prussin
- Charles E. Via, Jr. Department of Civil and Environmental Engineering, Virginia Tech, 1145 Perry Street, Blacksburg, VA 24061, United States
| | - Linsey C Marr
- Charles E. Via, Jr. Department of Civil and Environmental Engineering, Virginia Tech, 1145 Perry Street, Blacksburg, VA 24061, United States
| | - Lenwood S Heath
- Department of Computer Science, Virginia Tech, 225 Stranger Street, Blacksburg, VA 24061, United States
| | - Peter J Vikesland
- Charles E. Via, Jr. Department of Civil and Environmental Engineering, Virginia Tech, 1145 Perry Street, Blacksburg, VA 24061, United States
| | - Liqing Zhang
- Department of Computer Science, Virginia Tech, 225 Stranger Street, Blacksburg, VA 24061, United States
| | - Amy Pruden
- Charles E. Via, Jr. Department of Civil and Environmental Engineering, Virginia Tech, 1145 Perry Street, Blacksburg, VA 24061, United States.
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14
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Arumugam K, Bessarab I, Haryono MAS, Liu X, Zuniga-Montanez RE, Roy S, Qiu G, Drautz-Moses DI, Law YY, Wuertz S, Lauro FM, Huson DH, Williams RBH. Recovery of complete genomes and non-chromosomal replicons from activated sludge enrichment microbial communities with long read metagenome sequencing. NPJ Biofilms Microbiomes 2021; 7:23. [PMID: 33727564 PMCID: PMC7966762 DOI: 10.1038/s41522-021-00196-6] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2020] [Accepted: 02/12/2021] [Indexed: 01/31/2023] Open
Abstract
New long read sequencing technologies offer huge potential for effective recovery of complete, closed genomes from complex microbial communities. Using long read data (ONT MinION) obtained from an ensemble of activated sludge enrichment bioreactors we recover 22 closed or complete genomes of community members, including several species known to play key functional roles in wastewater bioprocesses, specifically microbes known to exhibit the polyphosphate- and glycogen-accumulating organism phenotypes (namely Candidatus Accumulibacter and Dechloromonas, and Micropruina, Defluviicoccus and Candidatus Contendobacter, respectively), and filamentous bacteria (Thiothrix) associated with the formation and stability of activated sludge flocs. Additionally we demonstrate the recovery of close to 100 circularised plasmids, phages and small microbial genomes from these microbial communities using long read assembled sequence. We describe methods for validating long read assembled genomes using their counterpart short read metagenome-assembled genomes, and assess the influence of different correction procedures on genome quality and predicted gene quality. Our findings establish the feasibility of performing long read metagenome-assembled genome recovery for both chromosomal and non-chromosomal replicons, and demonstrate the value of parallel sampling of moderately complex enrichment communities to obtaining high quality reference genomes of key functional species relevant for wastewater bioprocesses.
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Affiliation(s)
- Krithika Arumugam
- Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, Singapore, Singapore
| | - Irina Bessarab
- Singapore Centre for Environmental Life Sciences Engineering, National University of Singapore, Singapore, Singapore
| | - Mindia A S Haryono
- Singapore Centre for Environmental Life Sciences Engineering, National University of Singapore, Singapore, Singapore
| | - Xianghui Liu
- Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, Singapore, Singapore
| | - Rogelio E Zuniga-Montanez
- Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, Singapore, Singapore
- Department of Civil and Environmental Engineering, One Shields Avenue, University of California, Davis, CA, USA
| | - Samarpita Roy
- Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, Singapore, Singapore
| | - Guanglei Qiu
- Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, Singapore, Singapore
- School of Environment and Energy, South China University of Technology, Guangzhou, China
| | - Daniela I Drautz-Moses
- Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, Singapore, Singapore
| | - Ying Yu Law
- Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, Singapore, Singapore
| | - Stefan Wuertz
- Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, Singapore, Singapore
- School of Civil and Environmental Engineering, Nanyang Technological University, Singapore, Singapore
| | - Federico M Lauro
- Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, Singapore, Singapore
- Asian School of the Environment, Nanyang Technological University, Singapore, Singapore
| | - Daniel H Huson
- Institute for Bioinformatics and Medical Informatics, University of Tübingen, Tübingen, Germany
- Life Sciences Institute, National University of Singapore, Singapore, Singapore
| | - Rohan B H Williams
- Singapore Centre for Environmental Life Sciences Engineering, National University of Singapore, Singapore, Singapore.
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15
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Filling the Gaps in the Cyanobacterial Tree of Life-Metagenome Analysis of Stigonema ocellatum DSM 106950, Chlorogloea purpurea SAG 13.99 and Gomphosphaeria aponina DSM 107014. Genes (Basel) 2021; 12:genes12030389. [PMID: 33803228 PMCID: PMC8001431 DOI: 10.3390/genes12030389] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2020] [Revised: 02/18/2021] [Accepted: 03/03/2021] [Indexed: 12/26/2022] Open
Abstract
Cyanobacteria represent one of the most important and diverse lineages of prokaryotes with an unparalleled morphological diversity ranging from unicellular cocci and characteristic colony-formers to multicellular filamentous strains with different cell types. Sequencing of more than 1200 available reference genomes was mainly driven by their ecological relevance (Prochlorococcus, Synechococcus), toxicity (Microcystis) and the availability of axenic strains. In the current study three slowly growing non-axenic cyanobacteria with a distant phylogenetic positioning were selected for metagenome sequencing in order to (i) investigate their genomes and to (ii) uncover the diversity of associated heterotrophs. High-throughput Illumina sequencing, metagenomic assembly and binning allowed us to establish nearly complete high-quality draft genomes of all three cyanobacteria and to determine their phylogenetic position. The cyanosphere of the limnic isolates comprises up to 40 heterotrophic bacteria that likely coexisted for several decades, and it is dominated by Alphaproteobacteria and Bacteriodetes. The diagnostic marker protein RpoB ensured in combination with our novel taxonomic assessment via BLASTN-dependent text-mining a reliable classification of the metagenome assembled genomes (MAGs). The detection of one new family and more than a dozen genera of uncultivated heterotrophic bacteria illustrates that non-axenic cyanobacteria are treasure troves of hidden microbial diversity.
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16
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Dreher TW, Davis EW, Mueller RS. Complete genomes derived by directly sequencing freshwater bloom populations emphasize the significance of the genus level ADA clade within the Nostocales. HARMFUL ALGAE 2021; 103:102005. [PMID: 33980445 DOI: 10.1016/j.hal.2021.102005] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/27/2020] [Revised: 02/20/2021] [Accepted: 02/27/2021] [Indexed: 06/12/2023]
Abstract
The genome sequences of 16 Nostocales cyanobacteria have been determined. Most of them are complete or near-complete genome sequences derived by long-read metagenome sequencing of recent harmful algal blooms (HABs) in freshwater lakes without the potential bias of culture isolation. The genomes are all members of the recently recognized ADA clade (Driscoll et al., Harmful Algae, 77:93, 2018), which we argue represents a genus. We identify 10 putative species-level branches within the clade, on the basis of 91-gene phylogenomic and average nucleotide identity analyses. The assembled genomes each correspond to a single morphotype in the original sample, but distinct genomes from different HABs in some cases correspond to similar morphotypes. We present data indicating that the ADA clade is a highly significant component of current cyanobacterial HABs, including members assigned to the prevalent Dolichospermum and Aphanizomenon genera, as well as Cuspidothrix and Anabaena. In general, currently used genus and species names within the ADA clade are not monophyletic. We infer that the morphological characters routinely used in taxonomic assignments are not reliable for discriminating species within the ADA clade. Taxonomic revisions will be needed to create a genus with a single name (we recommend Anabaena) and to adopt species names that do not depend on morphological traits that lack sufficient discrimination and specificity, while recognizing the utility of some easily observable and distinct morphologies.
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Affiliation(s)
- Theo W Dreher
- Department of Microbiology, Oregon State University, 226 Nash Hall, Corvallis, OR 97331, USA; Center for Genome Research and Biocomputing, Oregon State University, Corvallis, OR 97331, USA.
| | - Edward W Davis
- Center for Genome Research and Biocomputing, Oregon State University, Corvallis, OR 97331, USA
| | - Ryan S Mueller
- Department of Microbiology, Oregon State University, 226 Nash Hall, Corvallis, OR 97331, USA
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17
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Brown CL, Keenum IM, Dai D, Zhang L, Vikesland PJ, Pruden A. Critical evaluation of short, long, and hybrid assembly for contextual analysis of antibiotic resistance genes in complex environmental metagenomes. Sci Rep 2021; 11:3753. [PMID: 33580146 PMCID: PMC7881036 DOI: 10.1038/s41598-021-83081-8] [Citation(s) in RCA: 47] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2020] [Accepted: 01/05/2021] [Indexed: 02/06/2023] Open
Abstract
In the fight to limit the global spread of antibiotic resistance, the assembly of environmental metagenomes has the potential to provide rich contextual information (e.g., taxonomic hosts, carriage on mobile genetic elements) about antibiotic resistance genes (ARG) in the environment. However, computational challenges associated with assembly can impact the accuracy of downstream analyses. This work critically evaluates the impact of assembly leveraging short reads, nanopore MinION long-reads, and a combination of the two (hybrid) on ARG contextualization for ten environmental metagenomes using seven prominent assemblers (IDBA-UD, MEGAHIT, Canu, Flye, Opera-MS, metaSpades and HybridSpades). While short-read and hybrid assemblies produced similar patterns of ARG contextualization, raw or assembled long nanopore reads produced distinct patterns. Based on an in-silico spike-in experiment using real and simulated reads, we show that low to intermediate coverage species are more likely to be incorporated into chimeric contigs across all assemblers and sequencing technologies, while more abundant species produce assemblies with a greater frequency of inversions and insertion/deletions (indels). In sum, our analyses support hybrid assembly as a valuable technique for boosting the reliability and accuracy of assembly-based analyses of ARGs and neighboring genes at environmentally-relevant coverages, provided that sufficient short-read sequencing depth is achieved.
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Affiliation(s)
- Connor L Brown
- Genetics, Bioinformatics, and Computational Biology, Virginia Tech, Blacksburg, VA, 24060, USA
| | - Ishi M Keenum
- Department of Civil & Environmental Engineering, Virginia Tech, Blacksburg, VA, 24060, USA
| | - Dongjuan Dai
- Department of Civil & Environmental Engineering, Virginia Tech, Blacksburg, VA, 24060, USA
| | - Liqing Zhang
- Genetics, Bioinformatics, and Computational Biology, Virginia Tech, Blacksburg, VA, 24060, USA.
| | - Peter J Vikesland
- Department of Civil & Environmental Engineering, Virginia Tech, Blacksburg, VA, 24060, USA
| | - Amy Pruden
- Department of Civil & Environmental Engineering, Virginia Tech, Blacksburg, VA, 24060, USA.
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18
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Van Damme R, Hölzer M, Viehweger A, Müller B, Bongcam-Rudloff E, Brandt C. Metagenomics workflow for hybrid assembly, differential coverage binning, metatranscriptomics and pathway analysis (MUFFIN). PLoS Comput Biol 2021; 17:e1008716. [PMID: 33561126 PMCID: PMC7899367 DOI: 10.1371/journal.pcbi.1008716] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2020] [Revised: 02/22/2021] [Accepted: 01/17/2021] [Indexed: 01/12/2023] Open
Abstract
Metagenomics has redefined many areas of microbiology. However, metagenome-assembled genomes (MAGs) are often fragmented, primarily when sequencing was performed with short reads. Recent long-read sequencing technologies promise to improve genome reconstruction. However, the integration of two different sequencing modalities makes downstream analyses complex. We, therefore, developed MUFFIN, a complete metagenomic workflow that uses short and long reads to produce high-quality bins and their annotations. The workflow is written by using Nextflow, a workflow orchestration software, to achieve high reproducibility and fast and straightforward use. This workflow also produces the taxonomic classification and KEGG pathways of the bins and can be further used for quantification and annotation by providing RNA-Seq data (optionally). We tested the workflow using twenty biogas reactor samples and assessed the capacity of MUFFIN to process and output relevant files needed to analyze the microbial community and their function. MUFFIN produces functional pathway predictions and, if provided de novo metatranscript annotations across the metagenomic sample and for each bin. MUFFIN is available on github under GNUv3 licence: https://github.com/RVanDamme/MUFFIN.
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Affiliation(s)
- Renaud Van Damme
- Department of Molecular Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden
- Department Animal Breeding and Genetics, Bioinformatics section, Swedish University of Agricultural Sciences, Uppsala, Sweden
| | - Martin Hölzer
- RNA Bioinformatics and High-Throughput Analysis, Friedrich Schiller University Jena, Jena, Germany
| | - Adrian Viehweger
- RNA Bioinformatics and High-Throughput Analysis, Friedrich Schiller University Jena, Jena, Germany
- Department of Medical Microbiology, University Hospital Leipzig, Leipzig Germany
| | - Bettina Müller
- Department of Molecular Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden
| | - Erik Bongcam-Rudloff
- Department Animal Breeding and Genetics, Bioinformatics section, Swedish University of Agricultural Sciences, Uppsala, Sweden
| | - Christian Brandt
- Department Animal Breeding and Genetics, Bioinformatics section, Swedish University of Agricultural Sciences, Uppsala, Sweden
- Institute for Infectious Diseases and Infection Control, Jena University Hospital, Jena, Germany
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19
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Bharti R, Grimm DG. Current challenges and best-practice protocols for microbiome analysis. Brief Bioinform 2021; 22:178-193. [PMID: 31848574 PMCID: PMC7820839 DOI: 10.1093/bib/bbz155] [Citation(s) in RCA: 284] [Impact Index Per Article: 71.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2019] [Revised: 10/23/2019] [Accepted: 11/06/2019] [Indexed: 12/15/2022] Open
Abstract
Analyzing the microbiome of diverse species and environments using next-generation sequencing techniques has significantly enhanced our understanding on metabolic, physiological and ecological roles of environmental microorganisms. However, the analysis of the microbiome is affected by experimental conditions (e.g. sequencing errors and genomic repeats) and computationally intensive and cumbersome downstream analysis (e.g. quality control, assembly, binning and statistical analyses). Moreover, the introduction of new sequencing technologies and protocols led to a flood of new methodologies, which also have an immediate effect on the results of the analyses. The aim of this work is to review the most important workflows for 16S rRNA sequencing and shotgun and long-read metagenomics, as well as to provide best-practice protocols on experimental design, sample processing, sequencing, assembly, binning, annotation and visualization. To simplify and standardize the computational analysis, we provide a set of best-practice workflows for 16S rRNA and metagenomic sequencing data (available at https://github.com/grimmlab/MicrobiomeBestPracticeReview).
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Affiliation(s)
- Richa Bharti
- Weihenstephan-Triesdorf University of Applied Sciences and Technical University of Munich, TUM Campus Straubing for Biotechnology and Sustainability, Straubing, Germany
| | - Dominik G Grimm
- Weihenstephan-Triesdorf University of Applied Sciences and Technical University of Munich, TUM Campus Straubing for Biotechnology and Sustainability, Straubing, Germany
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20
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Olson ND, Treangen TJ, Hill CM, Cepeda-Espinoza V, Ghurye J, Koren S, Pop M. Metagenomic assembly through the lens of validation: recent advances in assessing and improving the quality of genomes assembled from metagenomes. Brief Bioinform 2020; 20:1140-1150. [PMID: 28968737 DOI: 10.1093/bib/bbx098] [Citation(s) in RCA: 86] [Impact Index Per Article: 17.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2017] [Revised: 07/13/2017] [Indexed: 01/09/2023] Open
Abstract
Metagenomic samples are snapshots of complex ecosystems at work. They comprise hundreds of known and unknown species, contain multiple strain variants and vary greatly within and across environments. Many microbes found in microbial communities are not easily grown in culture making their DNA sequence our only clue into their evolutionary history and biological function. Metagenomic assembly is a computational process aimed at reconstructing genes and genomes from metagenomic mixtures. Current methods have made significant strides in reconstructing DNA segments comprising operons, tandem gene arrays and syntenic blocks. Shorter, higher-throughput sequencing technologies have become the de facto standard in the field. Sequencers are now able to generate billions of short reads in only a few days. Multiple metagenomic assembly strategies, pipelines and assemblers have appeared in recent years. Owing to the inherent complexity of metagenome assembly, regardless of the assembly algorithm and sequencing method, metagenome assemblies contain errors. Recent developments in assembly validation tools have played a pivotal role in improving metagenomics assemblers. Here, we survey recent progress in the field of metagenomic assembly, provide an overview of key approaches for genomic and metagenomic assembly validation and demonstrate the insights that can be derived from assemblies through the use of assembly validation strategies. We also discuss the potential for impact of long-read technologies in metagenomics. We conclude with a discussion of future challenges and opportunities in the field of metagenomic assembly and validation.
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21
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Levy Karin E, Mirdita M, Söding J. MetaEuk-sensitive, high-throughput gene discovery, and annotation for large-scale eukaryotic metagenomics. MICROBIOME 2020; 8:48. [PMID: 32245390 PMCID: PMC7126354 DOI: 10.1186/s40168-020-00808-x] [Citation(s) in RCA: 128] [Impact Index Per Article: 25.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/15/2019] [Accepted: 02/14/2020] [Indexed: 05/10/2023]
Abstract
BACKGROUND Metagenomics is revolutionizing the study of microorganisms and their involvement in biological, biomedical, and geochemical processes, allowing us to investigate by direct sequencing a tremendous diversity of organisms without the need for prior cultivation. Unicellular eukaryotes play essential roles in most microbial communities as chief predators, decomposers, phototrophs, bacterial hosts, symbionts, and parasites to plants and animals. Investigating their roles is therefore of great interest to ecology, biotechnology, human health, and evolution. However, the generally lower sequencing coverage, their more complex gene and genome architectures, and a lack of eukaryote-specific experimental and computational procedures have kept them on the sidelines of metagenomics. RESULTS MetaEuk is a toolkit for high-throughput, reference-based discovery, and annotation of protein-coding genes in eukaryotic metagenomic contigs. It performs fast searches with 6-frame-translated fragments covering all possible exons and optimally combines matches into multi-exon proteins. We used a benchmark of seven diverse, annotated genomes to show that MetaEuk is highly sensitive even under conditions of low sequence similarity to the reference database. To demonstrate MetaEuk's power to discover novel eukaryotic proteins in large-scale metagenomic data, we assembled contigs from 912 samples of the Tara Oceans project. MetaEuk predicted >12,000,000 protein-coding genes in 8 days on ten 16-core servers. Most of the discovered proteins are highly diverged from known proteins and originate from very sparsely sampled eukaryotic supergroups. CONCLUSION The open-source (GPLv3) MetaEuk software (https://github.com/soedinglab/metaeuk) enables large-scale eukaryotic metagenomics through reference-based, sensitive taxonomic and functional annotation. Video abstract.
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Affiliation(s)
- Eli Levy Karin
- Quantitative and Computational Biology, Max-Planck Institute for Biophysical Chemistry, 37077, Göttingen, Germany.
| | - Milot Mirdita
- Quantitative and Computational Biology, Max-Planck Institute for Biophysical Chemistry, 37077, Göttingen, Germany
| | - Johannes Söding
- Quantitative and Computational Biology, Max-Planck Institute for Biophysical Chemistry, 37077, Göttingen, Germany.
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Chen LX, Anantharaman K, Shaiber A, Eren AM, Banfield JF. Accurate and complete genomes from metagenomes. Genome Res 2020; 30:315-333. [PMID: 32188701 PMCID: PMC7111523 DOI: 10.1101/gr.258640.119] [Citation(s) in RCA: 223] [Impact Index Per Article: 44.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Genomes are an integral component of the biological information about an organism; thus, the more complete the genome, the more informative it is. Historically, bacterial and archaeal genomes were reconstructed from pure (monoclonal) cultures, and the first reported sequences were manually curated to completion. However, the bottleneck imposed by the requirement for isolates precluded genomic insights for the vast majority of microbial life. Shotgun sequencing of microbial communities, referred to initially as community genomics and subsequently as genome-resolved metagenomics, can circumvent this limitation by obtaining metagenome-assembled genomes (MAGs); but gaps, local assembly errors, chimeras, and contamination by fragments from other genomes limit the value of these genomes. Here, we discuss genome curation to improve and, in some cases, achieve complete (circularized, no gaps) MAGs (CMAGs). To date, few CMAGs have been generated, although notably some are from very complex systems such as soil and sediment. Through analysis of about 7000 published complete bacterial isolate genomes, we verify the value of cumulative GC skew in combination with other metrics to establish bacterial genome sequence accuracy. The analysis of cumulative GC skew identified potential misassemblies in some reference genomes of isolated bacteria and the repeat sequences that likely gave rise to them. We discuss methods that could be implemented in bioinformatic approaches for curation to ensure that metabolic and evolutionary analyses can be based on very high-quality genomes.
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Affiliation(s)
- Lin-Xing Chen
- Department of Earth and Planetary Sciences, University of California, Berkeley, California 94720, USA
| | - Karthik Anantharaman
- Department of Earth and Planetary Sciences, University of California, Berkeley, California 94720, USA
| | - Alon Shaiber
- Graduate Program in Biophysical Sciences, University of Chicago, Chicago, Illinois 60637, USA.,Department of Medicine, University of Chicago, Chicago, Illinois 60637, USA
| | - A Murat Eren
- Department of Medicine, University of Chicago, Chicago, Illinois 60637, USA.,Bay Paul Center, Marine Biological Laboratory, Woods Hole, Massachusetts 02543, USA
| | - Jillian F Banfield
- Department of Earth and Planetary Sciences, University of California, Berkeley, California 94720, USA.,Department of Environmental Science, Policy, and Management, University of California, Berkeley, California 94720, USA.,Earth and Environmental Sciences, Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720, USA
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Aziz A, Currie BJ, Mayo M, Sarovich DS, Price EP. Comparative genomics confirms a rare melioidosis human-to-human transmission event and reveals incorrect phylogenomic reconstruction due to polyclonality. Microb Genom 2020; 6:e000326. [PMID: 31958055 PMCID: PMC7067207 DOI: 10.1099/mgen.0.000326] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2019] [Accepted: 12/18/2019] [Indexed: 01/13/2023] Open
Abstract
Human-to-human transmission of the melioidosis bacterium, Burkholderia pseudomallei, is exceedingly rare, with only a handful of suspected cases documented to date. Here, we used whole-genome sequencing (WGS) to characterize one such unusual B. pseudomallei transmission event, which occurred between a breastfeeding mother with mastitis and her child. Two strains corresponding to multilocus sequence types (STs)-259 and -261 were identified in the mother's sputum from both the primary culture sweep and in purified colonies, confirming an unusual polyclonal infection in this patient. In contrast, primary culture sweeps of the mother's breast milk and the child's cerebrospinal fluid and blood samples contained only ST-259, indicating monoclonal transmission to the child. Analysis of purified ST-259 isolates showed no genetic variation between mother and baby isolates, providing the strongest possible evidence of B. pseudomallei human-to-human transmission, probably via breastfeeding. Next, phylogenomic analysis of all isolates, including the mother's mixed ST-259/ST-261 sputum sample, was performed to investigate the effects of mixtures on phylogenetic inference. Inclusion of this mixture caused a dramatic reduction in the number of informative SNPs, resulting in branch collapse of ST-259 and ST-261 isolates, and several instances of incorrect topology in a global B. pseudomallei phylogeny, resulting in phylogenetic incongruence. Although phylogenomics can provide clues about the presence of mixtures within WGS datasets, our results demonstrate that this methodology can lead to phylogenetic misinterpretation if mixed genomes are not correctly identified and omitted. Using current bioinformatic tools, we demonstrate a robust method for bacterial mixture identification and strain parsing that avoids these pitfalls.
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Affiliation(s)
- Ammar Aziz
- Global and Tropical Health Division, Menzies School of Health Research, Charles Darwin University, Darwin, NT, Australia
| | - Bart J. Currie
- Global and Tropical Health Division, Menzies School of Health Research, Charles Darwin University, Darwin, NT, Australia
- Infectious Diseases Department, Royal Darwin Hospital, Darwin, NT, Australia
| | - Mark Mayo
- Global and Tropical Health Division, Menzies School of Health Research, Charles Darwin University, Darwin, NT, Australia
| | - Derek S. Sarovich
- Global and Tropical Health Division, Menzies School of Health Research, Charles Darwin University, Darwin, NT, Australia
- GeneCology Research Centre, University of the Sunshine Coast, Sippy Downs, QLD, Australia
- Sunshine Coast Health Institute, Birtinya, QLD, Australia
| | - Erin P. Price
- Global and Tropical Health Division, Menzies School of Health Research, Charles Darwin University, Darwin, NT, Australia
- GeneCology Research Centre, University of the Sunshine Coast, Sippy Downs, QLD, Australia
- Sunshine Coast Health Institute, Birtinya, QLD, Australia
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24
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Chan AWY, Naphtali J, Schellhorn HE. High-throughput DNA sequencing technologies for water and wastewater analysis. Sci Prog 2019; 102:351-376. [PMID: 31818206 PMCID: PMC10424514 DOI: 10.1177/0036850419881855] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Conventional microbiological water monitoring uses culture-dependent techniques to screen indicator microbial species such as Escherichia coli and fecal coliforms. With high-throughput, second-generation sequencing technologies becoming less expensive, water quality monitoring programs can now leverage the massively parallel nature of second-generation sequencing technologies for batch sample processing to simultaneously obtain compositional and functional information of culturable and as yet uncultured microbial organisms. This review provides an introduction to the technical capabilities and considerations necessary for the use of second-generation sequencing technologies, specifically 16S rDNA amplicon and whole-metagenome sequencing, to investigate the composition and functional potential of microbiomes found in water and wastewater systems.
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Affiliation(s)
| | - James Naphtali
- Department of Biology, McMaster University, Hamilton, ON, Canada
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25
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Breitwieser FP, Lu J, Salzberg SL. A review of methods and databases for metagenomic classification and assembly. Brief Bioinform 2019; 20:1125-1136. [PMID: 29028872 PMCID: PMC6781581 DOI: 10.1093/bib/bbx120] [Citation(s) in RCA: 294] [Impact Index Per Article: 49.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2017] [Revised: 08/22/2017] [Indexed: 12/13/2022] Open
Abstract
Microbiome research has grown rapidly over the past decade, with a proliferation of new methods that seek to make sense of large, complex data sets. Here, we survey two of the primary types of methods for analyzing microbiome data: read classification and metagenomic assembly, and we review some of the challenges facing these methods. All of the methods rely on public genome databases, and we also discuss the content of these databases and how their quality has a direct impact on our ability to interpret a microbiome sample.
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Affiliation(s)
| | | | - Steven L Salzberg
- Corresponding author: Steven L. Salzberg, Center for Computational Biology, Johns Hopkins University, 1900 E. Monument St., Baltimore, MD, 21205, USA. E-mail:
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26
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Abstract
Microbiome research has grown rapidly over the past decade, with a proliferation of new methods that seek to make sense of large, complex data sets. Here, we survey two of the primary types of methods for analyzing microbiome data: read classification and metagenomic assembly, and we review some of the challenges facing these methods. All of the methods rely on public genome databases, and we also discuss the content of these databases and how their quality has a direct impact on our ability to interpret a microbiome sample.
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27
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Boolchandani M, D'Souza AW, Dantas G. Sequencing-based methods and resources to study antimicrobial resistance. Nat Rev Genet 2019; 20:356-370. [PMID: 30886350 PMCID: PMC6525649 DOI: 10.1038/s41576-019-0108-4] [Citation(s) in RCA: 207] [Impact Index Per Article: 34.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Antimicrobial resistance extracts high morbidity, mortality and economic costs yearly by rendering bacteria immune to antibiotics. Identifying and understanding antimicrobial resistance are imperative for clinical practice to treat resistant infections and for public health efforts to limit the spread of resistance. Technologies such as next-generation sequencing are expanding our abilities to detect and study antimicrobial resistance. This Review provides a detailed overview of antimicrobial resistance identification and characterization methods, from traditional antimicrobial susceptibility testing to recent deep-learning methods. We focus on sequencing-based resistance discovery and discuss tools and databases used in antimicrobial resistance studies.
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Affiliation(s)
- Manish Boolchandani
- The Edison Family Center for Genome Sciences and Systems Biology, Washington University in St. Louis School of Medicine, St. Louis, MO, USA
| | - Alaric W D'Souza
- The Edison Family Center for Genome Sciences and Systems Biology, Washington University in St. Louis School of Medicine, St. Louis, MO, USA
| | - Gautam Dantas
- The Edison Family Center for Genome Sciences and Systems Biology, Washington University in St. Louis School of Medicine, St. Louis, MO, USA.
- Department of Pathology & Immunology, Washington University in St. Louis School of Medicine, St. Louis, MO, USA.
- Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO, USA.
- Department of Molecular Microbiology, Washington University in St. Louis School of Medicine, St. Louis, MO, USA.
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Warwick-Dugdale J, Solonenko N, Moore K, Chittick L, Gregory AC, Allen MJ, Sullivan MB, Temperton B. Long-read viral metagenomics captures abundant and microdiverse viral populations and their niche-defining genomic islands. PeerJ 2019; 7:e6800. [PMID: 31086738 PMCID: PMC6487183 DOI: 10.7717/peerj.6800] [Citation(s) in RCA: 81] [Impact Index Per Article: 13.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2018] [Accepted: 03/14/2019] [Indexed: 01/18/2023] Open
Abstract
Marine viruses impact global biogeochemical cycles via their influence on host community structure and function, yet our understanding of viral ecology is constrained by limitations in host culturing and a lack of reference genomes and 'universal' gene markers to facilitate community surveys. Short-read viral metagenomic studies have provided clues to viral function and first estimates of global viral gene abundance and distribution, but their assemblies are confounded by populations with high levels of strain evenness and nucleotide diversity (microdiversity), limiting assembly of some of the most abundant viruses on Earth. Such features also challenge assembly across genomic islands containing niche-defining genes that drive ecological speciation. These populations and features may be successfully captured by single-virus genomics and fosmid-based approaches, at least in abundant taxa, but at considerable cost and technical expertise. Here we established a low-cost, low-input, high throughput alternative sequencing and informatics workflow to improve viral metagenomic assemblies using short-read and long-read technology. The 'VirION' (Viral, long-read metagenomics via MinION sequencing) approach was first validated using mock communities where it was found to be as relatively quantitative as short-read methods and provided significant improvements in recovery of viral genomes. We then then applied VirION to the first metagenome from a natural viral community from the Western English Channel. In comparison to a short-read only approach, VirION: (i) increased number and completeness of assembled viral genomes; (ii) captured abundant, highly microdiverse virus populations, and (iii) captured more and longer genomic islands. Together, these findings suggest that VirION provides a high throughput and cost-effective alternative to fosmid and single-virus genomic approaches to more comprehensively explore viral communities in nature.
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Affiliation(s)
- Joanna Warwick-Dugdale
- Plymouth Marine Laboratory, Plymouth, Devon, United Kingdom
- School of Biosciences, University of Exeter, Exeter, Devon, United Kingdom
| | - Natalie Solonenko
- Department of Microbiology, Ohio State University, Columbus, OH, United States of America
| | - Karen Moore
- School of Biosciences, University of Exeter, Exeter, Devon, United Kingdom
| | - Lauren Chittick
- Department of Microbiology, Ohio State University, Columbus, OH, United States of America
| | - Ann C. Gregory
- Department of Microbiology, Ohio State University, Columbus, OH, United States of America
| | - Michael J. Allen
- Plymouth Marine Laboratory, Plymouth, Devon, United Kingdom
- School of Biosciences, University of Exeter, Exeter, Devon, United Kingdom
| | - Matthew B. Sullivan
- Department of Microbiology, Ohio State University, Columbus, OH, United States of America
- Civil, Environmental and Geodetic Engineering, Ohio State University, Columbus, OH, United States of America
| | - Ben Temperton
- School of Biosciences, University of Exeter, Exeter, Devon, United Kingdom
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Culturomics of the plant prokaryotic microbiome and the dawn of plant-based culture media - A review. J Adv Res 2019; 19:15-27. [PMID: 31341666 PMCID: PMC6630032 DOI: 10.1016/j.jare.2019.04.002] [Citation(s) in RCA: 71] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2019] [Revised: 04/11/2019] [Accepted: 04/12/2019] [Indexed: 12/22/2022] Open
Abstract
The plant microbiome culturomics is substantially lagging behind the human microbiome. Conventional chemically-synthetic culture media recover < 10% of plant-associated microbiota. Plant-based culture media (PCM) are introduced as a novel tool for plant microbiome culturomics. PCM extended the microbiota culturability to recover unculturable bacterial taxa. Streamlined- and large-genomes conspicuously contribute to the dilemma of unculturability. Improving cultivability of a wider range of bacterial and archaeal community members, living natively in natural environments and within plants, is a prerequisite to better understanding plant-microbiota interactions and their functions in such very complex systems. Sequencing, assembling, and annotation of pure microbial strain genomes provide higher quality data compared to environmental metagenome analyses, and can substantially improve gene and protein database information. Despite the comprehensive knowledge which already was gained using metagenomic and metatranscriptomic methods, there still exists a big gap in understanding in vivo microbial gene functioning in planta, since many differentially expressed genes or gene families are not yet annotated. Here, the progress in culturing procedures for plant microbiota depending on plant-based culture media, and their proficiency in obtaining single prokaryotic isolates of novel and rapidly increasing candidate phyla are reviewed. As well, the great success of culturomics of the human microbiota is considered with the main objective of encouraging microbiologists to continue minimizing the gap between the microbial richness in nature and the number of species in culture, for the benefit of both basic and applied microbiology. The clear message to fellow plant microbiologists is to apply plant-tailored culturomic techniques that might open up novel procedures to obtain not-yet-cultured organisms and extend the known plant microbiota repertoire to unprecedented levels.
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Uritskiy G, DiRuggiero J. Applying Genome-Resolved Metagenomics to Deconvolute the Halophilic Microbiome. Genes (Basel) 2019; 10:genes10030220. [PMID: 30875864 PMCID: PMC6471235 DOI: 10.3390/genes10030220] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2019] [Revised: 03/08/2019] [Accepted: 03/11/2019] [Indexed: 12/25/2022] Open
Abstract
In the past decades, the study of microbial life through shotgun metagenomic sequencing has rapidly expanded our understanding of environmental, synthetic, and clinical microbial communities. Here, we review how shotgun metagenomics has affected the field of halophilic microbial ecology, including functional potential reconstruction, virus–host interactions, pathway selection, strain dispersal, and novel genome discoveries. However, there still remain pitfalls and limitations from conventional metagenomic analysis being applied to halophilic microbial communities. Deconvolution of halophilic metagenomes has been difficult due to the high G + C content of these microbiomes and their high intraspecific diversity, which has made both metagenomic assembly and binning a challenge. Halophiles are also underrepresented in public genome databases, which in turn slows progress. With this in mind, this review proposes experimental and analytical strategies to overcome the challenges specific to the halophilic microbiome, from experimental designs to data acquisition and the computational analysis of metagenomic sequences. Finally, we speculate about the potential applications of other next-generation sequencing technologies in halophilic communities. RNA sequencing, long-read technologies, and chromosome conformation assays, not initially intended for microbiomes, are becoming available in the study of microbial communities. Together with recent analytical advancements, these new methods and technologies have the potential to rapidly advance the field of halophile research.
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Affiliation(s)
- Gherman Uritskiy
- Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA.
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31
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Lambrechts S, Willems A, Tahon G. Uncovering the Uncultivated Majority in Antarctic Soils: Toward a Synergistic Approach. Front Microbiol 2019; 10:242. [PMID: 30828325 PMCID: PMC6385771 DOI: 10.3389/fmicb.2019.00242] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2018] [Accepted: 01/29/2019] [Indexed: 01/22/2023] Open
Abstract
Although Antarctica was once believed to be a sterile environment, it is now clear that the microbial communities inhabiting the Antarctic continent are surprisingly diverse. Until the beginning of the new millennium, little was known about the most abundant inhabitants of the continent: prokaryotes. From then on, however, the rising use of deep sequencing techniques has led to a better understanding of the Antarctic prokaryote diversity and provided insights in the composition of prokaryotic communities in different Antarctic environments. Although these cultivation-independent approaches can produce millions of sequences, linking these data to organisms is hindered by several problems. The largest difficulty is the lack of biological information on large parts of the microbial tree of life, arising from the fact that most microbial diversity on Earth has never been characterized in laboratory cultures. These unknown prokaryotes, also known as microbial dark matter, have been dominantly detected in all major environments on our planet. Laboratory cultures provide access to the complete genome and the means to experimentally verify genomic predictions and metabolic functions and to provide evidence of horizontal gene transfer. Without such well-documented reference data, microbial dark matter will remain a major blind spot in deep sequencing studies. Here, we review our current understanding of prokaryotic communities in Antarctic ice-free soils based on cultivation-dependent and cultivation-independent approaches. We discuss advantages and disadvantages of both approaches and how these strategies may be combined synergistically to strengthen each other and allow a more profound understanding of prokaryotic life on the frozen continent.
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Affiliation(s)
- Sam Lambrechts
- Laboratory of Microbiology, Department of Biochemistry and Microbiology, Ghent University, Ghent, Belgium
| | | | - Guillaume Tahon
- Laboratory of Microbiology, Department of Biochemistry and Microbiology, Ghent University, Ghent, Belgium
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NPBSS: a new PacBio sequencing simulator for generating the continuous long reads with an empirical model. BMC Bioinformatics 2018; 19:177. [PMID: 29788930 PMCID: PMC5964698 DOI: 10.1186/s12859-018-2208-0] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2018] [Accepted: 05/15/2018] [Indexed: 12/29/2022] Open
Abstract
Background PacBio sequencing platform offers longer read lengths than the second-generation sequencing technologies. It has revolutionized de novo genome assembly and enabled the automated reconstruction of reference-quality genomes. Due to its extremely wide range of application areas, fast sequencing simulation systems with high fidelity are in great demand to facilitate the development and comparison of subsequent analysis tools. Although there are several available simulators (e.g., PBSIM, SimLoRD and FASTQSim) that target the specific generation of PacBio libraries, the error rate of simulated sequences is not well matched to the quality value of raw PacBio datasets, especially for PacBio’s continuous long reads (CLR). Results By analyzing the characteristic features of CLR data from PacBio SMRT (single molecule real time) sequencing, we developed a new PacBio sequencing simulator (called NPBSS) for producing CLR reads. NPBSS simulator firstly samples the read sequences according to the read length logarithmic normal distribution, and choses different base quality values with different proportions. Then, NPBSS computes the overall error probability of each base in the read sequence with an empirical model, and calculates the deletion, substitution and insertion probabilities with the overall error probability to generate the PacBio CLR reads. Alignment results demonstrate that NPBSS fits the error rate of the PacBio CLR reads better than PBSIM and FASTQSim. In addition, the assembly results also show that simulated sequences of NPBSS are more like real PacBio CLR data. Conclusion NPBSS simulator is convenient to use with efficient computation and flexible parameters setting. Its generating PacBio CLR reads are more like real PacBio datasets. Electronic supplementary material The online version of this article (10.1186/s12859-018-2208-0) contains supplementary material, which is available to authorized users.
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Cabello-Yeves PJ, Zemskaya TI, Rosselli R, Coutinho FH, Zakharenko AS, Blinov VV, Rodriguez-Valera F. Genomes of Novel Microbial Lineages Assembled from the Sub-Ice Waters of Lake Baikal. Appl Environ Microbiol 2018; 84:e02132-17. [PMID: 29079621 PMCID: PMC5734018 DOI: 10.1128/aem.02132-17] [Citation(s) in RCA: 55] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2017] [Accepted: 10/19/2017] [Indexed: 11/20/2022] Open
Abstract
We present a metagenomic study of Lake Baikal (East Siberia). Two samples obtained from the water column under the ice cover (5 and 20 m deep) in March 2016 have been deep sequenced and the reads assembled to generate metagenome-assembled genomes (MAGs) that are representative of the microbes living in this special environment. Compared with freshwater bodies studied around the world, Lake Baikal had an unusually high fraction of Verrucomicrobia Other groups, such as Actinobacteria and Proteobacteria, were in proportions similar to those found in other lakes. The genomes (and probably cells) tended to be small, presumably reflecting the extremely oligotrophic and cold prevalent conditions. Baikal microbes are novel lineages recruiting very little from other water bodies and are distantly related to other freshwater microbes. Despite their novelty, they showed the closest relationship to genomes discovered by similar approaches from other freshwater lakes and reservoirs. Some of them were particularly similar to MAGs from the Baltic Sea, which, although it is brackish, connected to the ocean, and much more eutrophic, has similar climatological conditions. Many of the microbes contained rhodopsin genes, indicating that, in spite of the decreased light penetration allowed by the thick ice/snow cover, photoheterotrophy could be widespread in the water column, either because enough light penetrates or because the microbes are already adapted to the summer ice-less conditions. We have found a freshwater SAR11 subtype I/II representative showing striking synteny with Pelagibacterubique strains, as well as a phage infecting the widespread freshwater bacterium PolynucleobacterIMPORTANCE Despite the increasing number of metagenomic studies on different freshwater bodies, there is still a missing component in oligotrophic cold lakes suffering from long seasonal frozen cycles. Here, we describe microbial genomes from metagenomic assemblies that appear in the upper water column of Lake Baikal, the largest and deepest freshwater body on Earth. This lake is frozen from January to May, which generates conditions that include an inverted temperature gradient (colder up), decrease in light penetration due to ice, and, especially, snow cover, and oligotrophic conditions more similar to the open-ocean and high-altitude lakes than to other freshwater or brackish systems. As could be expected, most reconstructed genomes are novel lineages distantly related to others in cold environments, like the Baltic Sea and other freshwater lakes. Among them, there was a broad set of streamlined microbes with small genomes/intergenic spacers, including a new nonmarine Pelagibacter-like (subtype I/II) genome.
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Affiliation(s)
- Pedro J Cabello-Yeves
- Evolutionary Genomics Group, Departamento de Producción Vegetal y Microbiología, Universidad Miguel Hernández, San Juan de Alicante, Alicante, Spain
| | - Tamara I Zemskaya
- Limnological Institute, Siberian Branch of the Russian Academy of Sciences, Irkutsk, Russia
| | - Riccardo Rosselli
- Evolutionary Genomics Group, Departamento de Producción Vegetal y Microbiología, Universidad Miguel Hernández, San Juan de Alicante, Alicante, Spain
| | - Felipe H Coutinho
- Evolutionary Genomics Group, Departamento de Producción Vegetal y Microbiología, Universidad Miguel Hernández, San Juan de Alicante, Alicante, Spain
| | - Alexandra S Zakharenko
- Limnological Institute, Siberian Branch of the Russian Academy of Sciences, Irkutsk, Russia
| | - Vadim V Blinov
- Limnological Institute, Siberian Branch of the Russian Academy of Sciences, Irkutsk, Russia
| | - Francisco Rodriguez-Valera
- Evolutionary Genomics Group, Departamento de Producción Vegetal y Microbiología, Universidad Miguel Hernández, San Juan de Alicante, Alicante, Spain
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Li F, Neves ALA, Ghoshal B, Guan LL. Symposium review: Mining metagenomic and metatranscriptomic data for clues about microbial metabolic functions in ruminants. J Dairy Sci 2017; 101:5605-5618. [PMID: 29274958 DOI: 10.3168/jds.2017-13356] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2017] [Accepted: 10/27/2017] [Indexed: 12/22/2022]
Abstract
Metagenomics and metatranscriptomics can capture the whole genome and transcriptome repertoire of microorganisms through sequencing total DNA/RNA from various environmental samples, providing both taxonomic and functional information with high resolution. The unique and complex rumen microbial ecosystem is receiving great research attention because the rumen microbiota coevolves with the host and equips ruminants with the ability to convert cellulosic plant materials to high-protein products for human consumption. To date, hundreds to thousands of microbial phylotypes have been identified in the rumen using culture-independent molecular-based approaches, and genomic information of rumen microorganisms is rapidly accumulating through the single genome sequencing. However, functional characteristics of the rumen microbiome have not been well described because there are numerous uncultivable microorganisms in the rumen. The advent of metagenomics and metatranscriptomics along with advanced bioinformatics methods can help us better understand mechanisms of the rumen fermentation, which is vital for improving nutrient utilization and animal productivity. Therefore, in this review, we summarize a general workflow to conduct rumen metagenomics and metatranscriptomics and discuss how the data can be interpreted to be useful information. Moreover, we review recent literatures studying associations between the rumen microbiome and host phenotypes (e.g., feed efficiency and methane emissions) using these approaches, aiming to provide a useful guide to include studying the rumen microbiome as one of the research objectives using these 2 approaches.
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Affiliation(s)
- Fuyong Li
- Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5
| | - Andre L A Neves
- Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5
| | - Bibaswan Ghoshal
- Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5
| | - Le Luo Guan
- Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5.
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