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Targeted recovery of novel phylogenetic diversity from next-generation sequence data.

Lynch MD, Bartram AK, Neufeld JD - ISME J (2012)

Bottom Line: We combined BLASTN network analysis, phylogenetics and targeted primer design to amplify 16S rRNA gene sequences from unique potential bacterial lineages, comprising part of the rare biosphere from a multi-million sequence data set from an Arctic tundra soil sample.Demonstrating the feasibility of the protocol developed here, three of seven recovered phylogenetic lineages represented extremely divergent taxonomic entities.A comparison to twelve next-generation data sets from additional soils suggested persistent low-abundance distributions of these novel 16S rRNA genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Waterloo, Waterloo, ON, Canada.

ABSTRACT
Next-generation sequencing technologies have led to recognition of a so-called 'rare biosphere'. These microbial operational taxonomic units (OTUs) are defined by low relative abundance and may be specifically adapted to maintaining low population sizes. We hypothesized that mining of low-abundance next-generation 16S ribosomal RNA (rRNA) gene data would lead to the discovery of novel phylogenetic diversity, reflecting microorganisms not yet discovered by previous sampling efforts. Here, we test this hypothesis by combining molecular and bioinformatic approaches for targeted retrieval of phylogenetic novelty within rare biosphere OTUs. We combined BLASTN network analysis, phylogenetics and targeted primer design to amplify 16S rRNA gene sequences from unique potential bacterial lineages, comprising part of the rare biosphere from a multi-million sequence data set from an Arctic tundra soil sample. Demonstrating the feasibility of the protocol developed here, three of seven recovered phylogenetic lineages represented extremely divergent taxonomic entities. These divergent target sequences correspond to (a) a previously unknown lineage within the BRC1 candidate phylum, (b) a sister group to the early diverging and currently recognized monospecific Cyanobacteria Gloeobacter, a genus containing multiple plesiomorphic traits and (c) a highly divergent lineage phylogenetically resolved within mitochondria. A comparison to twelve next-generation data sets from additional soils suggested persistent low-abundance distributions of these novel 16S rRNA genes. The results demonstrate this sequence analysis and retrieval pipeline as applicable for exploring underrepresented phylogenetic novelty and recovering taxa that may represent significant steps in bacterial evolution.

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Related in: MedlinePlus

Subtrees (a–f) of experimental UL sequences from Figure 3 demonstrating local phylogenetic patterns. Node support values correspond to maximum likelihood parametric bootstrap scores ⩾50%.*=all subsequent branch lengths are 50% to scale.
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fig4: Subtrees (a–f) of experimental UL sequences from Figure 3 demonstrating local phylogenetic patterns. Node support values correspond to maximum likelihood parametric bootstrap scores ⩾50%.*=all subsequent branch lengths are 50% to scale.

Mentions: Sequences from UL4 grouped in several clades within the Bacteroidetes and all classified to the Sphingobacteriales. Half of the 12 sequences formed a sister clade relative to Nubsella zeaxanthinifaciens (Figure 4a) and showed highest identity with Pedobacter sp. in BLASTN analysis. Three of the remaining sequences appeared to be phylogenetically novel, sister to, but divergent from aquatic bacterial species Microscilla marina and Flexibacter elegans (Figure 4b). This topology was not well supported by bootstrap analysis, although the clade's position within the larger group was well supported. Similar to UL4, sequences from UL6 predominantly grouped in three areas of the tree; however, six of 11 sequences grouped diffusely throughout the Planctomycetales (Figure 4c). Four of the remaining sequences grouped strongly within the Chloroflexi, but could not be assigned to more specific taxonomic ranks (Figure 4d).


Targeted recovery of novel phylogenetic diversity from next-generation sequence data.

Lynch MD, Bartram AK, Neufeld JD - ISME J (2012)

Subtrees (a–f) of experimental UL sequences from Figure 3 demonstrating local phylogenetic patterns. Node support values correspond to maximum likelihood parametric bootstrap scores ⩾50%.*=all subsequent branch lengths are 50% to scale.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475379&req=5

fig4: Subtrees (a–f) of experimental UL sequences from Figure 3 demonstrating local phylogenetic patterns. Node support values correspond to maximum likelihood parametric bootstrap scores ⩾50%.*=all subsequent branch lengths are 50% to scale.
Mentions: Sequences from UL4 grouped in several clades within the Bacteroidetes and all classified to the Sphingobacteriales. Half of the 12 sequences formed a sister clade relative to Nubsella zeaxanthinifaciens (Figure 4a) and showed highest identity with Pedobacter sp. in BLASTN analysis. Three of the remaining sequences appeared to be phylogenetically novel, sister to, but divergent from aquatic bacterial species Microscilla marina and Flexibacter elegans (Figure 4b). This topology was not well supported by bootstrap analysis, although the clade's position within the larger group was well supported. Similar to UL4, sequences from UL6 predominantly grouped in three areas of the tree; however, six of 11 sequences grouped diffusely throughout the Planctomycetales (Figure 4c). Four of the remaining sequences grouped strongly within the Chloroflexi, but could not be assigned to more specific taxonomic ranks (Figure 4d).

Bottom Line: We combined BLASTN network analysis, phylogenetics and targeted primer design to amplify 16S rRNA gene sequences from unique potential bacterial lineages, comprising part of the rare biosphere from a multi-million sequence data set from an Arctic tundra soil sample.Demonstrating the feasibility of the protocol developed here, three of seven recovered phylogenetic lineages represented extremely divergent taxonomic entities.A comparison to twelve next-generation data sets from additional soils suggested persistent low-abundance distributions of these novel 16S rRNA genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Waterloo, Waterloo, ON, Canada.

ABSTRACT
Next-generation sequencing technologies have led to recognition of a so-called 'rare biosphere'. These microbial operational taxonomic units (OTUs) are defined by low relative abundance and may be specifically adapted to maintaining low population sizes. We hypothesized that mining of low-abundance next-generation 16S ribosomal RNA (rRNA) gene data would lead to the discovery of novel phylogenetic diversity, reflecting microorganisms not yet discovered by previous sampling efforts. Here, we test this hypothesis by combining molecular and bioinformatic approaches for targeted retrieval of phylogenetic novelty within rare biosphere OTUs. We combined BLASTN network analysis, phylogenetics and targeted primer design to amplify 16S rRNA gene sequences from unique potential bacterial lineages, comprising part of the rare biosphere from a multi-million sequence data set from an Arctic tundra soil sample. Demonstrating the feasibility of the protocol developed here, three of seven recovered phylogenetic lineages represented extremely divergent taxonomic entities. These divergent target sequences correspond to (a) a previously unknown lineage within the BRC1 candidate phylum, (b) a sister group to the early diverging and currently recognized monospecific Cyanobacteria Gloeobacter, a genus containing multiple plesiomorphic traits and (c) a highly divergent lineage phylogenetically resolved within mitochondria. A comparison to twelve next-generation data sets from additional soils suggested persistent low-abundance distributions of these novel 16S rRNA genes. The results demonstrate this sequence analysis and retrieval pipeline as applicable for exploring underrepresented phylogenetic novelty and recovering taxa that may represent significant steps in bacterial evolution.

Show MeSH
Related in: MedlinePlus