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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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Maximum Likelihood phylogeny constructed from nearly full-length 16S rRNA gene sequences. Sequences amplified from putative novel V3 sequences combined with seed sequences of known taxonomy derived from the Living Tree Project.
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fig3: Maximum Likelihood phylogeny constructed from nearly full-length 16S rRNA gene sequences. Sequences amplified from putative novel V3 sequences combined with seed sequences of known taxonomy derived from the Living Tree Project.

Mentions: Of the seven UL sequence sets amplified in this study, three were highly divergent from known bacterial lineages, representing significant, novel phylogenetic entities within the Cyanobacteria (UL9) and the BRC1 (UL5) candidate phylum, as well as a divergent group within the mitochondrial clade (UL13). Large species-rich bacterial phyla, such as the Firmicutes and various Proteobacteria, typically did not contain UL sequences (Figure 3). One exception was sequences retrieved with primers corresponding to the highly divergent UL13 clade, which resolved within the Alphaproteobacteria. Sequences from each targeted UL tended to be monophyletic (Figure 3), with the primary exception being sequences from UL11, which were broadly distributed throughout the phylogeny. One custom primer, UL14, did not successfully amplify product from the Alert soils and was therefore not represented in Sanger sequencing. This primer was designed against a clade of V3 sequences that resolved as sister to Clostridium taxa (Supplementary Figure S2).


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

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

Maximum Likelihood phylogeny constructed from nearly full-length 16S rRNA gene sequences. Sequences amplified from putative novel V3 sequences combined with seed sequences of known taxonomy derived from the Living Tree Project.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Maximum Likelihood phylogeny constructed from nearly full-length 16S rRNA gene sequences. Sequences amplified from putative novel V3 sequences combined with seed sequences of known taxonomy derived from the Living Tree Project.
Mentions: Of the seven UL sequence sets amplified in this study, three were highly divergent from known bacterial lineages, representing significant, novel phylogenetic entities within the Cyanobacteria (UL9) and the BRC1 (UL5) candidate phylum, as well as a divergent group within the mitochondrial clade (UL13). Large species-rich bacterial phyla, such as the Firmicutes and various Proteobacteria, typically did not contain UL sequences (Figure 3). One exception was sequences retrieved with primers corresponding to the highly divergent UL13 clade, which resolved within the Alphaproteobacteria. Sequences from each targeted UL tended to be monophyletic (Figure 3), with the primary exception being sequences from UL11, which were broadly distributed throughout the phylogeny. One custom primer, UL14, did not successfully amplify product from the Alert soils and was therefore not represented in Sanger sequencing. This primer was designed against a clade of V3 sequences that resolved as sister to Clostridium taxa (Supplementary Figure S2).

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