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A pipeline for targeted metagenomics of environmental bacteria.

ABSTRACT: BACKGROUND:Metagenomics and single cell genomics provide a window into the genetic repertoire of yet uncultivated microorganisms, but both methods are usually taxonomically untargeted. The combination of fluorescence in situ hybridization (FISH) and fluorescence activated cell sorting (FACS) has the potential to enrich taxonomically well-defined clades for genomic analyses. METHODS:Cells hybridized with a taxon-specific FISH probe are enriched based on their fluorescence signal via flow cytometric cell sorting. A recently developed FISH procedure, the hybridization chain reaction (HCR)-FISH, provides the high signal intensities required for flow cytometric sorting while maintaining the integrity of the cellular DNA for subsequent genome sequencing. Sorted cells are subjected to shotgun sequencing, resulting in targeted metagenomes of low diversity. RESULTS:Pure cultures of different taxonomic groups were used to (1) adapt and optimize the HCR-FISH protocol and (2) assess the effects of various cell fixation methods on both the signal intensity for cell sorting and the quality of subsequent genome amplification and sequencing. Best results were obtained for ethanol-fixed cells in terms of both HCR-FISH signal intensity and genome assembly quality. Our newly developed pipeline was successfully applied to a marine plankton sample from the North Sea yielding good quality metagenome assembled genomes from a yet uncultivated flavobacterial clade. CONCLUSIONS:With the developed pipeline, targeted metagenomes at various taxonomic levels can be efficiently retrieved from environmental samples. The resulting metagenome assembled genomes allow for the description of yet uncharacterized microbial clades. Video abstract.

SUBMITTER: Grieb A

PROVIDER: S-EPMC7024552 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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A pipeline for targeted metagenomics of environmental bacteria.

Grieb Anissa A Bowers Robert M RM Oggerin Monike M Goudeau Danielle D Lee Janey J Malmstrom Rex R RR Woyke Tanja T Fuchs Bernhard M BM

Microbiome 20200215 1

<h4>Background</h4>Metagenomics and single cell genomics provide a window into the genetic repertoire of yet uncultivated microorganisms, but both methods are usually taxonomically untargeted. The combination of fluorescence in situ hybridization (FISH) and fluorescence activated cell sorting (FACS) has the potential to enrich taxonomically well-defined clades for genomic analyses.<h4>Methods</h4>Cells hybridized with a taxon-specific FISH probe are enriched based on their fluorescence signal vi ...[more]

PMID: 32061258

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Project description:BackgroundThe production of 1 L of ethanol from sugarcane generates up to 12 L of vinasse, which is a liquid waste containing an as-yet uncharacterized microbial assemblage. Most vinasse is destined for use as a fertilizer on the sugarcane fields because of the high organic and K content; however, increased N2O emissions have been observed when vinasse is co-applied with inorganic N fertilizers. Here we aimed to characterize the microbial assemblage of vinasse to determine the gene potential of vinasse microbes for contributing to negative environmental effects during fertirrigation and/or to the obstruction of bioethanol fermentation.ResultsWe measured chemical characteristics and extracted total DNA from six vinasse batches taken over 1.5 years from a bioethanol and sugar mill in Sao Paulo State. The vinasse microbial assemblage was characterized by low alpha diversity with 5-15 species across the six vinasses. The core genus was Lactobacillus. The top six represented bacterial genera across the samples were Lactobacillus, Megasphaera and Mitsuokella (Phylum Firmicutes, 35-97% of sample reads); Arcobacter and Alcaligenes (Phylum Proteobacteria, 0-40%); Dysgonomonas (Phylum Bacteroidetes, 0-53%); and Bifidobacterium (Phylum Actinobacteria, 0-18%). Potential genes for denitrification but not nitrification were identified in the vinasse metagenomes, with putative nirK and nosZ genes the most represented. Binning resulted in 38 large bins with between 36.0 and 99.3% completeness, and five small mobile element bins. Of the large bins, 53% could be classified at the phylum level as Firmicutes, 15% as Proteobacteria, 13% as unknown phyla, 13% as Bacteroidetes and 6% as Actinobacteria. The large bins spanned a range of potential denitrifiers; moreover, the genetic repertoires of all the large bins included the presence of genes involved in acetate, CO2, ethanol, H2O2, and lactose metabolism; for many of the large bins, genes related to the metabolism of mannitol, xylose, butyric acid, cellulose, sucrose, "3-hydroxy" fatty acids and antibiotic resistance were present based on the annotations. In total, 21 vinasse bacterial draft genomes were submitted to the genome repository.ConclusionsIdentification of the gene repertoires of vinasse bacteria and assemblages supported the idea that organic carbon and nitrogen present in vinasse together with microbiological variation of vinasse might lead to varying patterns of N2O emissions during fertirrigation. Furthermore, we uncovered draft genomes of novel strains of known bioethanol contaminants, as well as draft genomes unknown at the phylum level. This study will aid efforts to improve bioethanol production efficiency and sugarcane agriculture sustainability.

Project description:BACKGROUND:Antimicrobial resistance is a major global health challenge. Metagenomics allows analyzing the presence and dynamics of "resistomes" (the ensemble of genes encoding antimicrobial resistance in a given microbiome) in disparate microbial ecosystems. However, the low sensitivity and specificity of available metagenomic methods preclude the detection of minority populations (often present below their detection threshold) and/or the identification of allelic variants that differ in the resulting phenotype. Here, we describe a novel strategy that combines targeted metagenomics using last generation in-solution capture platforms, with novel bioinformatics tools to establish a standardized framework that allows both quantitative and qualitative analyses of resistomes. METHODS:We developed ResCap, a targeted sequence capture platform based on SeqCapEZ (NimbleGene) technology, which includes probes for 8667 canonical resistance genes (7963 antibiotic resistance genes and 704 genes conferring resistance to metals or biocides), and 2517 relaxase genes (plasmid markers) and 78,600 genes homologous to the previous identified targets (47,806 for antibiotics and 30,794 for biocides or metals). Its performance was compared with metagenomic shotgun sequencing (MSS) for 17 fecal samples (9 humans, 8 swine). ResCap significantly improves MSS to detect "gene abundance" (from 2.0 to 83.2%) and "gene diversity" (26 versus 14.9 genes unequivocally detected per sample per million of reads; the number of reads unequivocally mapped increasing up to 300-fold by using ResCap), which were calculated using novel bioinformatic tools. ResCap also facilitated the analysis of novel genes potentially involved in the resistance to antibiotics, metals, biocides, or any combination thereof. CONCLUSIONS:ResCap, the first targeted sequence capture, specifically developed to analyze resistomes, greatly enhances the sensitivity and specificity of available metagenomic methods and offers the possibility to analyze genes related to the selection and transfer of antimicrobial resistance (biocides, heavy metals, plasmids). The model opens the possibility to study other complex microbial systems in which minority populations play a relevant role.

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A pipeline for targeted metagenomics of environmental bacteria.

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A pipeline for targeted metagenomics of environmental bacteria.

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