Design of targeted primers based on 16S rRNA sequences in meta-transcriptomic datasets and identification of a novel taxonomic group in the Asgard archaea

Zhang, Ruyi; Zou, Bin; Yan, Yonggang; Jeon, Che Ok; Li, Meng; Cai, Mingwei; Quan, Zhe‐Xue

doi:10.1186/s12866-020-1707-0

Cited by 14 publications

(9 citation statements)

References 59 publications

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“…Despite this recent focus on Asgardarchaeota, we have likely only explored a small fraction of the diversity encompassed by this phylum. Microbial community profiling based on small subunit (SSU) rRNA gene sequences suggest that many novel Asgardarchaeota lineages are awaiting genomic discovery 5,13,14 . Here we describe the recovery of 46 Asgardarchaeota MAGs from coastal, hot spring, and deep-sea sediments complemented by 25 MAGs extracted from public metagenomic datasets.…”

Section: Mainmentioning

confidence: 99%

Recoding enhances the metabolic capabilities of two novel methylotrophic Asgardarchaeota lineages

Sun

Evans

Gagen

et al. 2021

Preprint

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Asgardarchaeota have been proposed as the closest living relatives to eukaryotes, and a total of 72 metagenome-assembled genomes (MAGs) representing six primary lineages in this archaeal phylum have thus far been described. These organisms are predicted to be fermentative organoheterotrophs contributing to carbon cycling in sediment ecosystems. Here, we double the genomic catalogue of Asgardarchaeota by obtaining 71 MAGs from a range of habitats around the globe, including deep subsurface, shallow lake, and geothermal spring sediments. Phylogenomic inferences followed by taxonomic rank normalisation confirmed previously established Asgardarchaeota classes and revealed four novel lineages, two of which were consistently recovered as monophyletic classes. We therefore propose the names Candidatus Hodarchaeia class nov. and Cand. Jordarchaeia class nov., derived from the gods Hod and Jord in Norse mythology. Metabolic inference suggests that both novel classes represent methylotrophic acetogens, encoding the transfer of methyl groups, such as methylated amines, to coenzyme M with acetate as the end product in remnants of a methanogen-derived core metabolism. This inferred mode of energy conservation is predicted to be enhanced by genetic code expansions, i.e. recoding, allowing the incorporation of the rare 21st and 22nd amino acids selenocysteine (Sec) and pyrrolysine (Pyl). We found Sec recoding in Jordarchaeia and all other Asgardarchaeota classes, which likely benefit from increased catalytic activities of Sec-containing enzymes. Pyl recoding on the other hand is restricted to Hodarchaeia in the Asgardarchaeota, making it the first reported non-methanogenic lineage with an inferred complete Pyl machinery, likely providing this class with an efficient mechanism for methylamine utilisation. Furthermore, we identified enzymes for the biosynthesis of ester-type lipids, characteristic of Bacteria and Eukaryotes, in both novel classes, supporting the hypothesis that mixed ether-ester lipids are a shared feature among Asgardarchaeota.

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Section: Mainmentioning

confidence: 99%

Recoding enhances the metabolic capabilities of two novel methylotrophic Asgardarchaeota lineages

Sun

Evans

Gagen

et al. 2021

Preprint

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“…These ampliconbased approaches have been shown to reproduce the patterns of alpha and beta-diversity which are observed through shotgun sequencing [12]. However, amplicon methods can only detect organisms containing conserved sequences that are recognized by whatever PCR primers are chosen for the experiment; while there are several good choices available for bacterial 16S, eukaryotic 18S, and the Internal Transcribed Spacer (ITS), the development of improved primers is still an area of active research [17][18][19].…”

Section: Introductionmentioning

confidence: 99%

Universal Amplicon Sequencing of North Imperial Valley Wetlands Microbiomes

Becker¹,

Graham²,

Sager³

et al. 2022

Preprint

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DNA sequencing of complex microbial communities allows for the classification and quantification of thousands of distinct organisms in diverse environmental niches. We present a three domain "Universal Amplicon" (UA) method to simultaneously amplify DNA from the ribosomal small subunit locus from bacteria, archaea, and eukaryotes (and their organelles) using a single pair of amplification primers. We demonstrate the amenability of the UA to multiplexed Illumina library preparation and MiSeq-based sequencing. We validate the UA by sequencing a commercially available microbial community of known quantitative composition and through direct comparison to a shotgun metagenomics dataset. Following validation, we apply the UA to a time-course study of the wetlands of the Northern Imperial Valley in California and show substantial and variable microbial life in the Salton Sea and nearby waters. We demonstrate that the microbial ecology of the Salton Sea varies on at least a monthly basis and is distinct from the surrounding area. Finally, we contribute an open-source Shiny app for real-time analysis of complex metagenomic communities, with application to this study and far beyond.

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“…Either approach has limitations that are addressed by the other. Targeted PCR amplification can deeply sample microbial populations within a community, detecting even the rarest of members; how-ever, this approach may miss novel diversity by relying on previously sequenced genes for constructing PCR primers [8, 9, 10]. While every effort is made to ensure marker gene primers capture as much diversity as possible, amplification bias is always present [11].…”

Section: Introductionmentioning

confidence: 99%

PASV: Automatic protein partitioning and validation using conserved residues

Harrison

et al. 2021

Preprint

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Background: Increasingly, researchers use protein-coding genes from targeted PCR amplification or direct metagenomic sequencing in community and population ecology. Analysis of protein-coding genes presents different challenges from those encountered in traditional SSU rRNA studies. Most protein-coding sequences are annotated based on homology to other computationally-annotated sequences, which can lead to inaccurate annotations. Therefore, the results of sensitive homology searches must be validated to remove false-positives and assess functionality. Multiple lines of in silico evidence can be gathered by examining conserved domains and residues identified through biochemical investigations. However, manually validating sequences in this way can be time consuming and error prone, especially in large environmental studies. Results: An automated pipeline for protein active site validation (PASV) was developed to improve validation and partitioning accuracy for protein-coding sequences, combining multiple sequence alignment with expert domain knowledge. PASV was tested using commonly misannotated proteins: ribonucleotide reductase (RNR), alternative oxidase (AOX), and plastid terminal oxidase (PTOX). PASV partitioned 9,906 putative Class I alpha and Class II RNR sequences from bycatch in a global viral metagenomic investigation with >99% true positive and true negative rates. PASV predicted the class of 2,579 RNR sequences in >98% agreement with manual annotations. PASV correctly partitioned all 336 tested AOX and PTOX sequences. Conclusions: PASV provides an automated and accurate way to address post-homology search validation and partitioning of protein-coding marker genes. Source code is released under the MIT license and is found with documentation and usage examples on GitHub at https://github.com/mooreryan/pasv.

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Design of targeted primers based on 16S rRNA sequences in meta-transcriptomic datasets and identification of a novel taxonomic group in the Asgard archaea

Cited by 14 publications

References 59 publications

Recoding enhances the metabolic capabilities of two novel methylotrophic Asgardarchaeota lineages

Recoding enhances the metabolic capabilities of two novel methylotrophic Asgardarchaeota lineages

Universal Amplicon Sequencing of North Imperial Valley Wetlands Microbiomes

PASV: Automatic protein partitioning and validation using conserved residues

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