Connecting structure to function with the recovery of over 1000 high-quality activated sludge metagenome-assembled genomes encoding full-length rRNA genes using long-read sequencing

Singleton, Caitlin M.; Petriglieri, Francesca; Kristensen, Jannie Munk; Kirkegaard, Rasmus Hansen; Michaelsen, Thomas Yssing; Andersen, Martin Hjorth; Kondrotaite, Zivile; Karst, Søren Michael; Dueholm, Morten Simonsen; Nielsen, Per Halkjær; Albertsen, Mads

doi:10.1101/2020.05.12.088096

Cited by 27 publications

(28 citation statements)

References 91 publications

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“…The poor coverage likely relates to the fact that 16S rRNA genes often fail to assemble in MAGs produced by short-read sequencing data ( 32 ). This problem will likely disappear in the future with the introduction of more high-quality MAGs with complete rRNA genes into the GTDB as a result of long-read sequencing technologies, such as Nanopore and PacBio ( 33 , 34 ).…”

Section: Resultsmentioning

confidence: 99%

Generation of Comprehensive Ecosystem-Specific Reference Databases with Species-Level Resolution by High-Throughput Full-Length 16S rRNA Gene Sequencing and Automated Taxonomy Assignment (AutoTax)

Dueholm

Andersen

McIlroy

et al. 2020

mBio

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103

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High-throughput 16S rRNA gene amplicon sequencing is an essential method for studying the diversity and dynamics of microbial communities. However, this method is presently hampered by the lack of high-identity reference sequences for many environmental microbes in the public 16S rRNA gene reference databases and by the absence of a systematic and comprehensive taxonomy for the uncultured majority. Here, we demonstrate how high-throughput synthetic long-read sequencing can be applied to create ecosystem-specific full-length 16S rRNA gene amplicon sequence variant (FL-ASV) resolved reference databases that include high-identity references (>98.7% identity) for nearly all abundant bacteria (>0.01% relative abundance) using Danish wastewater treatment systems and anaerobic digesters as an example. In addition, we introduce a novel sequence identity-based approach for automated taxonomy assignment (AutoTax) that provides a complete seven-rank taxonomy for all reference sequences, using the SILVA taxonomy as a backbone, with stable placeholder names for unclassified taxa. The FL-ASVs are perfectly suited for the evaluation of taxonomic resolution and bias associated with primers commonly used for amplicon sequencing, allowing researchers to choose those that are ideal for their ecosystem. Reference databases processed with AutoTax greatly improves the classification of short-read 16S rRNA ASVs at the genus- and species-level, compared with the commonly used universal reference databases. Importantly, the placeholder names provide a way to explore the unclassified environmental taxa at different taxonomic ranks, which in combination with in situ analyses can be used to uncover their ecological roles.

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Section: Resultsmentioning

confidence: 99%

Generation of Comprehensive Ecosystem-Specific Reference Databases with Species-Level Resolution by High-Throughput Full-Length 16S rRNA Gene Sequencing and Automated Taxonomy Assignment (AutoTax)

Dueholm

Andersen

McIlroy

et al. 2020

mBio

Self Cite

103

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“…Recent advances in high-throughput sequencing methods have enabled the creation of comprehensive ecosystem-specific full-length 16S rRNA gene databases [25]. These databases can be used for detailed phylogenetic analyses, facilitating the design of specific fluorescence in situ hybridization (FISH) probes and re-evaluation of existing ones for the identification and characterization of key species [26]. Moreover, it is now possible to assemble high-quality, near-complete genomes from deeply sequenced metagenomes, revealing the metabolic potential of these organisms [26][27][28].…”

Section: Introductionmentioning

confidence: 99%

“…These databases can be used for detailed phylogenetic analyses, facilitating the design of specific fluorescence in situ hybridization (FISH) probes and re-evaluation of existing ones for the identification and characterization of key species [26]. Moreover, it is now possible to assemble high-quality, near-complete genomes from deeply sequenced metagenomes, revealing the metabolic potential of these organisms [26][27][28]. However, in situ validation of the genomic potential is critical and required to confirm their predicted role in the system.…”

Section: Introductionmentioning

confidence: 99%

“CandidatusDechloromonas phosphatis” and “CandidatusDechloromonas phosphovora”, two novel polyphosphate accumulating organisms abundant in wastewater treatment systems

Petriglieri

Singleton

Peces

et al. 2020

Preprint

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Members of the genus Dechloromonas are often abundant in enhanced biological phosphorus removal (EBPR) systems and are recognized putative polyphosphate accumulating organisms (PAOs), but their role in phosphate (P) removal is still unclear. Here, we used 16S rRNA gene sequencing and fluorescence in situ hybridization (FISH) to investigate the abundance and distribution of Dechloromonas spp. in Danish wastewater treatment plants. Two species were abundant, novel, and uncultured, and could be targeted by existing FISH probes. Raman microspectroscopy of probe-defined organisms (FISH-Raman) revealed the levels and dynamics of important intracellular storage polymers in abundant Dechloromonas spp. in the activated sludge from four full-scale EBPR plants and from a lab-scale sequencing batch reactor fed with different carbon sources (acetate, glucose, glycine, and glutamate). Moreover, 7 distinct Dechloromonas species were determined from a set of 10 high-quality metagenome-assembled genomes (MAGs) from Danish EBPR plants, each encoding the potential for poly-P, glycogen, and polyhydroxyalkanoates (PHA) accumulation. The two most abundant species exhibited an in situ phenotype in complete accordance with the metabolic information retrieved by the MAGs, with dynamic levels of poly-P, glycogen, and PHA during feast-famine anaerobic-aerobic cycling, legitimately placing these microorganisms among the important PAOs. As no isolates are available for the two species, we propose the names Candidatus Dechloromonas phosphatis and Candidatus Dechloromonas phosphovora.

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“…The most recent strategy in long-read metagenomics uses the long reads to obtain the raw metagenome assembly -ensuring the greatest contiguity of MAGs-and short reads to polish and improve the overall accuracy. This strategy was applied to assess the human GI microbiome [10], among others -such as mock communities [11], cow rumen [12], natural whey starter cultures [13] or wastewater [14]. Some authors suggest that we may overcome the need for short reads to polish long-read data by either using correction software, such as frameshift-aware correction [15] or with ultra-deep coverage of the genomes [11].…”

Section: Introductionmentioning

confidence: 99%

Long-Read Metagenomics to Retrieve High-Quality Metagenome-Assembled Genomes from Canine Feces

Cuscó¹,

Pérez²,

Viñes³

et al. 2020

Preprint

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Background. Metagenomics is a powerful and rapidly developing approach that provides new biological insights into the microbes inhabiting underexplored environments, such as canine fecal microbiome. We investigate long-read metagenomics with Nanopore sequencing to profile the fecal microbiome and to retrieve high-quality metagenome-assembled genomes (HQ MAGs) from a healthy dog.Results. More than 99% of total classified reads corresponded to Bacteria. The most abundant phylum was Bacteroidetes (~80% of total reads), followed by Firmicutes, Proteobacteria, and Fusobacteria. Prevotella (>50%) and Bacteroides (>20%) are the more abundant genera, followed by Fusobacterium, Megamonas, Sutterella, and other fecal-related genera, (each representing <5% of the total bacterial composition). We retrieved eight single-contig HQ MAGs and three medium-quality MAGs, after combining several metagenome dataset assemblies. The HQ MAGs corresponded to Succinivibrio, Sutterella, Prevotellamassilia, Phascolarctobacterium, Enterococcus, Blautia, and Catenibacterium genera. Succinivibrio HQ MAG represents a novel candidate bacterial species. Sutterella HQ MAG is potentially the first reported genome assembly for Sutterella stercoricanis, as assigned by 16S rRNA gene similarity. Prevotellamassilia, Phascolarctobacterium, Catenibacterium, and Blautia sp900541345 HQ MAGs improved the contiguity of previously reported genome assemblies in their respective genera, and the number of rRNA genes and tRNA genes. Finally, Enterococcus hirae and Blautia sp003287895 HQ MAGs represented species that already have a complete reference genome. At the technical level, we demonstrated that a high-molecular weight DNA extraction improved the taxonomic classification of the raw unassembled reads, the metagenomics assembly contiguity, and the retrieval of longer and circular contigs, which are potential HQ MAGs. Conclusions. Long-read metagenomics allowed us to recover HQ MAGs from canine feces of a healthy dog. The high-molecular weight DNA extraction to improve contiguity and the correction of the insertions and deletions to reduce frameshift errors ensure the retrieval of complete single-contig HQ MAGs.

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Connecting structure to function with the recovery of over 1000 high-quality activated sludge metagenome-assembled genomes encoding full-length rRNA genes using long-read sequencing

Cited by 27 publications

References 91 publications

Generation of Comprehensive Ecosystem-Specific Reference Databases with Species-Level Resolution by High-Throughput Full-Length 16S rRNA Gene Sequencing and Automated Taxonomy Assignment (AutoTax)

Generation of Comprehensive Ecosystem-Specific Reference Databases with Species-Level Resolution by High-Throughput Full-Length 16S rRNA Gene Sequencing and Automated Taxonomy Assignment (AutoTax)

“CandidatusDechloromonas phosphatis” and “CandidatusDechloromonas phosphovora”, two novel polyphosphate accumulating organisms abundant in wastewater treatment systems

Long-Read Metagenomics to Retrieve High-Quality Metagenome-Assembled Genomes from Canine Feces

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