Dataset Information

ORPER: A Workflow for Constrained SSU rRNA Phylogenies.

ABSTRACT: The continuous increase in sequenced genomes in public repositories makes the choice of interesting bacterial strains for future sequencing projects ever more complicated, as it is difficult to estimate the redundancy between these strains and the already available genomes. Therefore, we developed the Nextflow workflow "ORPER", for "ORganism PlacER", containerized in Singularity, which allows the determination the phylogenetic position of a collection of organisms in the genomic landscape. ORPER constrains the phylogenetic placement of SSU (16S) rRNA sequences in a multilocus reference tree based on ribosomal protein genes extracted from public genomes. We demonstrate the utility of ORPER on the Cyanobacteria phylum, by placing 152 strains of the BCCM/ULC collection.

SUBMITTER: Cornet L

PROVIDER: S-EPMC8623055 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature

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Publications

ORPER: A Workflow for Constrained SSU rRNA Phylogenies.

Cornet Luc L Ahn Anne-Catherine AC Wilmotte Annick A Baurain Denis D

Genes 20211029 11

The continuous increase in sequenced genomes in public repositories makes the choice of interesting bacterial strains for future sequencing projects ever more complicated, as it is difficult to estimate the redundancy between these strains and the already available genomes. Therefore, we developed the Nextflow workflow "ORPER", for "ORganism PlacER", containerized in Singularity, which allows the determination the phylogenetic position of a collection of organisms in the genomic landscape. ORPER ...[more]

PMID: 34828348

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Project description:BackgroundThe 16S rRNA gene is used extensively in bacterial phylogenetics, in species delineation, and now widely in microbiome studies. However, the gene suffers from intragenomic heterogeneity, and reports of recombination and an unreliable phylogenetic signal are accumulating. Here, we compare core gene phylogenies to phylogenies constructed using core gene concatenations to estimate the strength of signal for the 16S rRNA gene, its hypervariable regions, and all core genes at the intra- and inter-genus levels. Specifically, we perform four intra-genus analyses (Clostridium, n = 65; Legionella, n = 47; Staphylococcus, n = 36; and Campylobacter, n = 17) and one inter-genus analysis [41 core genera of the human gut microbiome (31 families, 17 orders, and 12 classes), n = 82].ResultsAt both taxonomic levels, the 16S rRNA gene was recombinant and subject to horizontal gene transfer. At the intra-genus level, the gene showed one of the lowest levels of concordance with the core genome phylogeny (50.7% average). Concordance for hypervariable regions was lower still, with entropy masking providing little to no benefit. A major factor influencing concordance was SNP count, which showed a positive logarithmic association. Using this relationship, we determined that 690 ± 110 SNPs were required for 80% concordance (average 16S rRNA gene SNP count was 254). We also found a wide range in 16S-23S-5S rRNA operon copy number among genomes (1-27). At the inter-genus level, concordance for the whole 16S rRNA gene was markedly higher (73.8% - 10th out of 49 loci); however, the most concordant hypervariable regions (V4, V3-V4, and V1-V2) ranked in the third quartile (62.5 to 60.0%).ConclusionsRamifications of a poor phylogenetic performance for the 16S rRNA gene are far reaching. For example, in addition to incorrect species/strain delineation and phylogenetic inference, it has the potential to confound community diversity metrics if phylogenetic information is incorporated - for example, with popular approaches such as Faith's phylogenetic diversity and UniFrac. Our results highlight the problematic nature of these approaches and their use (along with entropy masking) is discouraged. Lastly, the wide range in 16S rRNA gene copy number among genomes also has a strong potential to confound diversity metrics. Video Abstract.

Project description:Protists have traditionally been identified by cultivation and classified taxonomically based on their cellular morphologies and behavior. In the past decade, however, many novel protist taxa have been identified using cultivation independent ssu rRNA sequence surveys. New rRNA "phylotypes" from uncultivated eukaryotes have no connection to the wealth of prior morphological descriptions of protists. To link phylogenetically informative sequences with taxonomically informative morphological descriptions, we demonstrate several methods for combining whole cell rRNA-targeted fluorescent in situ hybridization (FISH) with cytoskeletal or organellar immunostaining. Either eukaryote or ciliate-specific ssu rRNA probes were combined with an anti-α-tubulin antibody or phalloidin, a common actin stain, to define cytoskeletal features of uncultivated protists in several environmental samples. The eukaryote ssu rRNA probe was also combined with Mitotracker® or a hydrogenosomal-specific anti-Hsp70 antibody to localize mitochondria and hydrogenosomes, respectively, in uncultivated protists from different environments. Using rRNA probes in combination with immunostaining, we linked ssu rRNA phylotypes with microtubule structure to describe flagellate and ciliate morphology in three diverse environments, and linked Naegleria spp. to their amoeboid morphology using actin staining in hay infusion samples. We also linked uncultivated ciliates to morphologically similar Colpoda-like ciliates using tubulin immunostaining with a ciliate-specific rRNA probe. Combining rRNA-targeted FISH with cytoskeletal immunostaining or stains targeting specific organelles provides a fast, efficient, high throughput method for linking genetic sequences with morphological features in uncultivated protists. When linked to phylotype, morphological descriptions of protists can both complement and vet the increasing number of sequences from uncultivated protists, including those of novel lineages, identified in diverse environments.

Dataset Information

ORPER: A Workflow for Constrained SSU rRNA Phylogenies.

Publications

ORPER: A Workflow for Constrained SSU rRNA Phylogenies.

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