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Sequencing the World of Possibilities for Energy & Environment
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Sequencing the World of Possibilities for Energy & Environment
Metagenomics with spatial resolution
When doing metagenomics, we take a sample from a
community and homogenise
The structure and complexityof a community are ignored
What about layers in microbial communities?
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Sequencing the World of Possibilities for Energy & Environment
Microelectrode measurements
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O2
H2S
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Introduction
• Hypersaline mat, Guerrero Negro, Baja California
– ~6cm thick
• The most diverse community on Earth
– Probably :)– Tens of thousands of microbial species– Dozens of novel phyla
• Steep physico/chemical gradients with depth
– O2, H2S, light
• Highly stratified (layered) community
– Cyanobacteria on top– Cloroflexi, proteobacteria,
bacteroides below
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Sequencing the World of Possibilities for Energy & Environment
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Sequencing the World of Possibilities for Energy & Environment
• Sequenced 10 layers of the mat
• Samples are taken: 1mm top layers, 3mm lower layers
• A parallel project is aimed to identify the diversity, deep sequencing of 16S is under way in the Norm Pace’ lab.
• Sequencing, from each layer:– ~13,000 reads (between 11,574 and 15,663)– Assembly gives contigs less than 10kb, all analysis
done with reads– ~13,600 proteins (between 12,012 and 15,984) – Loaded into IMG/M`
Sequencing strategy
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Sequencing the World of Possibilities for Energy & Environment
The questions
• How does the mat live?– Metabolism, interactions, chemotaxis
• Are there gradients observable from the data?– Species– Protein families– Functional families– Other characteristics?
• What is special about the mat as compared to other environments?
– Adaptations similar across many layers?– Adaptations that cross phylogenetic
boundaries?
• Stress management– Salinity– Heat
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Sequencing the World of Possibilities for Energy & Environment
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Depth (mm)
fraction of phylogenetic assignments per layer (%)
Unassigned Alphaproteobacteria
Cyanobacteria Deltaproteobacteria
Gammaproteobacteria Bacteroidetes
Blast
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Sequencing the World of Possibilities for Energy & Environment
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Depth (mm)
fraction of phylogenetic assignments per layer
Alphaproteobacteria Bacteriodetes Chloroflexi
Cyanobacteria Planctomycetes
Marker genes
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Sequencing the World of Possibilities for Energy & Environment
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Proteobacteria Bacteroidetes Chloroflexi Cyanobacteria Planctomycetes
Spirochaetes Verrucomicrobia
16S clone library
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Sequencing the World of Possibilities for Energy & Environment
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Proteobacteria Bacteroidetes Chloroflexi Cyanobacteria Planctomycetes
Spirochaetes Verrucomicrobia
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Depth (mm)
fraction of phylogenetic assignments per layer (%)
Unassigned Alphaproteobacteria
Cyanobacteria Deltaproteobacteria
Gammaproteobacteria Bacteroidetes
Species content - the discrepancies
16S clone library
Blast
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Depth (mm)
fraction of phylogenetic assignments per layer
Alphaproteobacteria Bacteriodetes Chloroflexi
Cyanobacteria Planctomycetes
Marker genes
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Sequencing the World of Possibilities for Energy & Environment
Functional content
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Sulfatases Methyltransferases Heterodisulfidereductase
ferredoxinoxidoreductase
DUF820 domain
Mat Gutless Worm Soil Whalefall Sludge AMD IMG
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Sequencing the World of Possibilities for Energy & Environment
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Sulfatases Methyltransferases Heterodisulfidereductase
ferredoxinoxidoreductase
DUF820 domain
Mat
Gutless Worm
Soil
Whalefall
Sludge
AMD
IMG
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Sequencing the World of Possibilities for Energy & Environment
Dep
th m
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O2
H2S
µM
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Sequencing the World of Possibilities for Energy & Environment
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Sequencing the World of Possibilities for Energy & Environment
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Sequencing the World of Possibilities for Energy & Environment
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Sequencing the World of Possibilities for Energy & Environment
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Sequencing the World of Possibilities for Energy & Environment
Unifying features?
• Layers are different– Phylogenetically– Functionally
• Anything common to all layers– Hypersalinity is common– Adaptations to hypersalinity?– K/Na exchange, increasing intracellular K
• In Archaea• Salinibacter Ruber• In the mat?
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Sequencing the World of Possibilities for Energy & Environment
Isoelectric profilesAverage isoelectric point distribution
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4.7 5.1 5.5 5.9 6.3 6.7 7.1 7.5 7.9 8.3 8.7 9.1 9.5 9.9
Isoelectric point
Fraction of dataset
Bacteria Archaea Mat Metagenomes Phages
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Sequencing the World of Possibilities for Energy & Environment
Amino acid usage
Aspartate content distribution
0102030405060708090
100
3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9
Proteomic aspartate fraction
Fraction of dataset
archaea bacteria mat microbiomes viruses
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Sequencing the World of Possibilities for Energy & Environment
GC content distribution of contigs in HSM
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GC contnent
Occurence
12345678910
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Sequencing the World of Possibilities for Energy & Environment
Conclusions
• Gradients found of– Phylogenetic groups– Protein families– Functional families
• Adaptations to salinity– Isoelectric point– Amino acid usage
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Sequencing the World of Possibilities for Energy & Environment
Acknowledgments
Jeroen RaesKirk Harris
Natalia Ivanova John Spear
Christian Von MeringBrad Bebout Peer Bork
Norman PacePhilip Hugenholtz