Connecting structure to function with the recovery of over 1000 high-quality metagenome-assembled genomes from activated sludge 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:10.1038/s41467-021-22203-2

Cited by 248 publications

(285 citation statements)

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“…The diversity of "Acidobacteriota" in the WWTPs was explored using the ecosystem-specific MiDAS 3 reference database and related MiDAS 3 taxonomy (Dueholm et al, 2020;Nierychlo et al, 2020), and MAGs. A total of 347 full-length 16S rRNA gene sequences belonging to "Acidobacteriota" were retrieved, 282 from the MiDAS 3 reference database, and 65 were from 50 high-quality MAGs obtained from Danish WWTPs (Singleton et al, 2021).…”

Section: Resultsmentioning

confidence: 99%

“…To determine how the 50 "Acidobacteriota" HQ MAGs, retrieved from a subset of the WWTPs investigated here (Singleton et al, 2021), compared to the diversity of described "Acidobacteriota" worldwide, we compared the 16S rRNA gene sequences to the SILVA138 Ref NR database (Supplementary Figure 1). The 16S rRNA gene sequences with genome classification from Genome Taxonomy Database (GTDB) (Parks et al, 2020) mostly had the same taxonomic classification as the SILVA138 Ref NR database.…”

Section: Resultsmentioning

confidence: 99%

“…"Acidobacteriota" MAGs from Singleton et al (2021) were classified taxonomically using GTDB-Tk (Chaumeil et al, 2019) v1.3.0, with species representatives determined using 95% average nucleotide clustering and dRep (Olm et al, 2017) v2.3…”

Section: Retrieval Of Genomes and Phylogenetic Classificationmentioning

confidence: 99%

“…Prokka v1.14 (Seemann, 2014) was also used to annotate the genomes and provide protein sequences to identify CXXCH motifs in multiheme cytochromes using a python script 4 . Infernal v1.1.2 (Nawrocki and Eddy, 2013) was used alongside BEDTools v2.27 (Quinlan and Hall, 2010) to retrieve the 16S rRNA genes for comparison to the MiDAS 16S rRNA gene sequences as in Singleton et al (2021). BLAST against NCBI nr was used to confirm genes of interest (Camacho et al, 2009).…”

Section: 2mentioning

confidence: 99%

“…Raman microspectroscopy was applied to characterize key physiological traits, such as presence of storage polymers in probe-defined members of abundant genera. In addition, we linked the fulllength 16S rRNA gene sequences to 50 high-quality MAGs, recently retrieved from the same WWTPs (Singleton et al, 2021), to provide insight into "Acidobacteriota" functional potential.…”

Section: Introductionmentioning

confidence: 99%

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High Diversity and Functional Potential of Undescribed “Acidobacteriota” in Danish Wastewater Treatment Plants

et al. 2021

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Microbial communities in water resource recovery facilities encompass a large diversity of poorly characterized lineages that could have undescribed process-critical functions. Recently, it was shown that taxa belonging to “Acidobacteriota” are abundant in Danish full-scale activated sludge wastewater treatment plants (WWTP), and here we investigated their diversity, distribution, and functional potential. “Acidobacteriota” taxa were identified using a comprehensive full-length 16S rRNA gene reference dataset and amplicon sequencing surveys across 37 WWTPs. Members of this phylum were diverse, belonging to 14 families, eight of which are completely uncharacterized and lack type strains. Several lineages were abundant, with relative abundances of up to 5% of the microbial community. Genome annotation and metabolic reconstruction of 50 high-quality “Acidobacteriota” metagenome-assembled genomes (MAGs) from 19 WWTPs showed high metabolic diversity and potential involvement in nitrogen and phosphorus removal and iron reduction. Fluorescence in situ hybridization (FISH) using newly-designed probes revealed cells with diverse morphologies, predominantly located inside activated sludge flocs. FISH in combination with Raman microspectroscopy revealed ecophysiological traits in probe-defined cells from the families Holophagaceae, Thermoanaerobaculaceae, and Vicinamibacteraceae, and families with the placeholder name of midas_f_502, midas_f_973, and midas_f_1548. Members of these lineages had the potential to be polyphosphate-accumulating organisms (PAOs) as intracellular storage was observed for the key compounds polyphosphate and glycogen.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Retrieval Of Genomes and Phylogenetic Classificationmentioning

confidence: 99%

Section: 2mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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High Diversity and Functional Potential of Undescribed “Acidobacteriota” in Danish Wastewater Treatment Plants

et al. 2021

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Selection of Biosurfactant‐Producing Microorganisms

Jaime

Romero

León

2023

Biosurfactants and Sustainability

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A Guide to Gene‐Centric Analysis Using TreeSAPP

Morgan-Lang

Hallam

2023

Current Protocols

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Gene-centric analysis is commonly used to chart the structure, function, and activity of microbial communities in natural and engineered environments. A common approach is to create custom ad hoc reference marker gene sets, but these come with the typical disadvantages of inaccuracy and limited utility beyond assigning query sequences taxonomic labels. The Tree-based Sensitive and Accurate Phylogenetic Profiler (TreeSAPP) software package standardizes analysis of phylogenetic and functional marker genes and improves predictive performance using a classification algorithm that leverages information-rich reference packages consisting of a multiple sequence alignment, a profile hidden Markov model, taxonomic lineage information, and a phylogenetic tree. Here, we provide a set of protocols that link the various analysis modules in TreeSAPP into a coherent process that both informs and directs the user experience. This workflow, initiated from a collection of candidate reference sequences, progresses through construction and refinement of a reference package to marker identification and normalized relative abundance calculations for homologous sequences in metagenomic and metatranscriptomic datasets. The alpha subunit of methyl-coenzyme M reductase (McrA) involved in biological methane cycling is presented as a use case given its dual role as a phylogenetic and functional marker gene driving an ecologically relevant process. These protocols fill several gaps in prior TreeSAPP documentation and provide best practices for reference package construction and refinement, including manual curation steps from trusted sources in support of reproducible gene-centric analysis.

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Connecting structure to function with the recovery of over 1000 high-quality metagenome-assembled genomes from activated sludge using long-read sequencing

Cited by 248 publications

References 100 publications

High Diversity and Functional Potential of Undescribed “Acidobacteriota” in Danish Wastewater Treatment Plants

High Diversity and Functional Potential of Undescribed “Acidobacteriota” in Danish Wastewater Treatment Plants

Selection of Biosurfactant‐Producing Microorganisms

A Guide to Gene‐Centric Analysis Using TreeSAPP

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