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FACE Soil Metagenome Comparisons in IMG. Melissa Dsouza , Peter Hallin , Craig Herbold, Rima Upchurch , & Paul Wilkinson . FACE Site Overview. Free-air and Carbon Dioxide Enrichment Elevated levels of CO2 expected in future decades Metagenome data in IMG/M Forested sites - PowerPoint PPT Presentation
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FACE Soil Metagenome Comparisons in IMG
Melissa Dsouza, Peter Hallin, Craig Herbold, Rima Upchurch, & Paul Wilkinson
FACE Site OverviewFree-air and Carbon Dioxide Enrichment
Elevated levels of CO2 expected in future decadesMetagenome data in IMG/M
Forested sites Duke (Sweetgum forest) Oak Ridge (Loblolly pine)
Nevada sites Soil crust Cresote
Maryland Estuary
Metagenomes OverviewSoil samplesStandardized extraction across all sites
Cheryl Kuske (LANL)FastDNA Spin kit2007-2008 sampling
Soil samplesExtracted using FastDNA Spin kit454 Titanium sequencing at JGI
Metagenomes OverviewSimilar GCSimilar
sequencing effortScaffold data
65-75% - 1 geneLength of
scaffolds varied low as 100 to 630 450-550 median
Basically unassembled reads
Questions about FACEQuestion 1: Are there differences in the taxonomic
and functional distributions across all sites?
Question 2: Are there are any differences between taxonomy within each elevated and ambient sites?
Question 3: Are there any functional differences within each elevated and ambient sites?
Taxonomy across sitesSimilar phyla present overall, but there were a few
differences between sites Actinobacteria and Proteobacteria top phyla
Swapped top or second between sites
Other top groups shifted between sitesFirmicutes, Bacteroidetes, Cyanobacteria,
Acidobacteria and Verrucomicrobia
Functional overview across sites
Nitrogen fixationAdded KEGG pathway (ko00910) to “Function
Card” and compared in Metagenome samplesOutput is matrix containing gene countsUsing R (Sorry IMG team!) we created a 2-
dimensional clustering.Clearly, samples cluster according to site, rather
than CO2 level.
Maryland Estuary CO +/- lights up
Clustering of sitesPCA of all COGs in all sitesAxes 1 and 2 explain ~90% of the variation
Largest variation in COGs appeared related to site rather than treatment
Remaining analyses done by site to examine differences by treatment
Desert
Estuary
Forests
Site Function SummaryDid not see significant
differences in 3 of 5 sitesMaryland Estuary
Membrane biogenesis-related functions
Soil crustDid not see a pattern
Was there overlap in COG functionsOne: a transposase in both
COG Pfam TIGRfamForest
Oak Ridge 0 0 0North Carolina 0 0 0
EstuaryMaryland 5 0 0
DesertSoil crust 7 8 2Creosote 0 0 0
Site Function SummaryDid not see significant
differences in 3 of 5 sitesMaryland Estuary
Membrane biogenesis-related functions
Soil crustDid not see a pattern
Was there overlap in COG functionsOne: a transposase in both
COG Pfam TIGRfamForest
Oak Ridge 0 0 0North Carolina 0 0 0
EstuaryMaryland 5 0 0
DesertSoil crust 7 8 2Creosote 0 0 0
TransposaseMaryland Estuary
Dominant group was Alphaproteobacteria
Nevada soil crust Dominant group was
Cyanobacteria
Reflective of dominant members of each site
Eurya
rchae
ota
Actino
bacter
ia
Chlor
oflex
i
Cyan
obact
eriaBa
cilli
Clostr
idia
Alpha
proteo
bacte
ria
Betap
roteo
bacter
ia
Epsilo
nprote
obact
eria
Gammap
roteo
bacte
riaThe
rmi
Gammap
roteo
bacter
ia0%2%4%6%8%
10%
Actino
bacte
ria
Alpha
proteo
bacte
riaBa
cilli
Betap
roteob
acteri
a
Chlor
oflexi
Cyan
obact
eria
Eurya
rchae
ota
Gammap
roteo
bacter
ia
Planct
omyce
tes
Strept
ophyta
0.00%2.00%4.00%6.00%8.00%
10.00%
Maryland EstuaryCOGs that were significant
Uncharacterized conserved proteinTransposaseGlycoltransferaseSerine/Theronine protein kinaseMembrane-fusion protein
Maryland EstuaryCOGs that were significant
Uncharacterized conserved proteinTransposaseGlycoltransferaseSerine/Theronine protein kinaseMembrane-fusion protein
Cell membrane biogenesisStudied phylogenetic distribution of the genes within
these COGs revealed…
Maryland EstuaryDeltaproteobacteria
were the dominant group for the phylogenetic distribution
This group might be the driving force between ambient and elevated within this site
Conclusion
Acidob
acteri
a
Actino
bacter
ia
Chlor
oflex
i
Planct
omyce
tes
Alphap
roteo
bacte
ria
Betap
roteo
bacte
ria
Deltap
roteo
bacter
ia0.00%2.00%4.00%6.00%8.00%
10.00%12.00%14.00%16.00%18.00%
Phylogenetic dis-trubution of genes
within COGS
Nevada Desert Soil CrustCOGs that were
significantAcetyltransferasesPredicted hydrolases
or acyltransferasesTransposaseCytochrome b subunit
of the bc complexPredicted ATPase FOG: PAS/PAC domainMolybdenum cofactor
biosynthesis enzyme
Pfams COX1 SBP_bac_5 HAMP Hydrolase MoeA_C DUF349 Radical_SAM SH3_3
TIGRfam YD repeat (two copies) delta-60 repeat domain
Nevada Desert Soil CrustCOGs that were
significantAcetyltransferasesPredicted hydrolases
or acyltransferasesTransposaseCytochrome b subunit
of the bc complexPredicted ATPase FOG: PAS/PAC domainMolybdenum cofactor
biosynthesis enzyme
Pfams COX1 SBP_bac_5 HAMP Hydrolase MoeA_C DUF349 Radical_SAM SH3_3
TIGRfam YD repeat (two copies) delta-60 repeat domain
Nevada Desert Soil CrustActinobacteria,
Alphaproteobacteria and Cyanobacteria were the most dominate in the distribution of functional categories
In one case Chloroflexi (shown right) were most dominate
ConclusionsAct
inobac
teria
Bacte
roide
tes
Chlor
oflex
i
Cyan
obact
eria
Clostr
idia
Fusob
acteri
a
Deltap
roteo
bacter
iaThe
rmi
Verru
comicr
obia
0.00%
0.50%
1.00%
1.50%
2.00%
2.50%
3.00%
3.50%Predicted ATPase
Final ConclusionsImpact of elevated carbon dioxide
Did not see any specific function changeTaxon played a major part in our analyses for
determining significance in functionsSequence quality
Our datasets were mainly unassembled sequencesLow quality (shorter reads) affects annotationFrame shift questions for 454 sequencingNot seeing the “real” picture of these sites