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Metagenomics:Principles and Applications
Raúl J. Cano, Ph.D.
Microorganisms in Soils
• Essential for life on Earth
• Vital for the formation and biogenesis of soil
• Maintain soil structure and fertility
• Central role in biogeochemical cycling processes
• Essential for plant growth (and bioremediation)
• Protect “host” from diseases
2
Essential for Sustainable Management of Soil Ecosystem Function, and Prediction of Impact
of Environmental Change
• Understanding the factors that determine:
– Growth, activity and diversity of soil microbial communities
– Their control by soil physicochemical characteristics and
– Interactions with other soil inhabitants
Carbonetto B, Rascovan N, Álvarez R, Mentaberry A, Vázquez MP (2014) Structure, Composition and Metagenomic Profile of Soil Microbiomes Associated to Agricultural Land Use and Tillage Systems in Argentine Pampas. PLoS ONE 9(6): e99949. doi:10.1371/journal.pone.0099949
Relative Abundances of Taxonomic Groups in Argentina Pampa Production Field Soil Microbiomes
3
Abundance is represented in terms of percentage in total bacterial sequences in a sample
Abundances of Different Orders in Bacteria in the 12 Sugar Beet Rhizosphere Samples.
4
The Rhizosphere Zoo
What is the “Microbiota”?
• The ecological community of commensal, mutualistic, and parasitic microorganisms that comprise an environment (e.g., soil, roots, gut)
5
Unraveling Environmental Process
Genomes in the Context of the EnvironmentResolution at the Species Level
• Genome sequence information provides the necessary foundation for subsequent field and lab analyses
• Mapping microbial community metabolism onto environmental processes
6
Strategies for Assessing Microbial Impact on Soil Quality and Plant Health
Defining Metagenomics
• The term “metagenome” was first used by J. Handelsman in 1998 to describe the sum total of the genetic material in an environmental sample
• Metagenomics combines molecular biology and genetics in an attempt to identify, and characterize the genetic material from (environmental) samples and apply that knowledge.
• The genetic diversity is assessed by isolation of DNA followed by direct analysis of the biota and corresponding functional genes from the sample
7
Why Metagenomics?
• Only a small proportion of organisms have been grown in culture
• Species do not live in isolation; they form communities
• Clonal cultures fail to represent the natural environment of a given organism
• Many proteins and protein functions remain undiscovered
Evolution of Sequencing Technologies
1980sSequencers were gel slabs using radioactive isotopsand thereafter using fluorescent chemistry (10 Kb/4 h)
1990s Capillary sequencers (50 Kb/h)
2005 Massive parallel pyrosequencing (20 MB/5 h)
2007 Sequencing by synthesis (1 GB/5 d)
2010 Single molecule sequencing (100 GB/5 d)
2013 Complete human genome in 15 min
8
The $100 Human Genome
Dynamics of Sequencing Costs
Cost of Sequencing has Driven Popularity of Metagenomics
SynthesisMethod
Read Length
Accuracy Reads/Run Time/Run Cost/mb
PGMIon Torrent
~ 400 bp 98%Up to 80 million
2 hours $1
Roche 454 Pyrosequencing
~ 700 bp 99.9% 1 million 24 hours $10
IlluminaSynthesis
50 – 300 bp
98% up to 3 billion 1‐10 days $0.05‐$0.15
SOLiDLigation
50+50 bp 99.9%1.2 to 1.4 billion
1‐2 weeks $0.13
SangerChain termination
~ 750 bp 99.9% N/A 0.3–3 hours $2,400
9
Basic Computational Tasks of Metagenomics
• Taxonomic analysis (who is out there?)
• Functional analysis (what are they doing?)
• Comparative analysis (how do they compare?)
Strategies for the Metagenomic Analysis of Environmental Microbial Communities
10
ASSESSING TAXONOMIC DIVERSITY
The Microbiome
What is the Microbiome
• Is the collection of ALL of the microbial genetic material found in a community of microbes living together
• It reflects the community structure of a particular environment
• Can be used to assess environmental changes and discriminate among various communities
11
How to Assess the Microbiome?
Small Subunit (SSU) Ribosomal RNA
• Present in all known life forms
• Highly conserved
• Can be used to differentiate among species
• Resistant to horizontal transfer events
SSU Ribosomal RNA
12
The PhyloChip
• The PhyloChip is a popular 16S rRNA gene microarray for microbial surveys
• Has been successfully used to study the microbial diversity of several interesting environments
• Its adoption, however, has been limited by a lack of accessible analysis software
Assessing and Analyzing the Microbiome
13
• Extract DNA and PCR‐amplify the target gene (or region)
— Usually SSU rRNA
• Each sample (aka microbiome) has its unique ID “barcode.”
• Known as “multiplex”
Combine and Sequence PCR Products (amplicons)
Workflow using SSU (16S/18S) Data
Sequences are NOT Directly Interpretable
Need to beware : – Quality of the
sequences
– Sequencing errors
– Chimeric PCR products
– Uneven sequence depth
– Short read effects
15
QIIME Overview
qualqual
fastafasta
MappingMapping
fastqfastq
fastQC_report
BIOM
TREE
alpha_rarefaction.pybeta_diversity_through_plots.pyjackknifed_beta_diversity.py
summarize_taxa_through_plots.py
Hue
coid
Sala
doid
Cont
empo
rary
hum
an s
tool
s
Actinomycetales
Bi dobacteriales
Bacteroidales
CytophagalesCytophagales
Bacillales
Lactobacillales
Clostridiales
Halanaerobiales
Thermoanaerobiales
Fusobacteriales
Rhizobiales
Rhodobacterales
Rhodospirillales
Burkholderiales
Sytrophobacteirales
Campylobactrales
Enterobacteriales
Legionellales
Methylococcales
Pseudomonadales
Vibrionales
Entomoplasmatales
Verrucomicrobiales
Saccharomycetales
Actiniaria
Coniferales
Lamiales
sffsff
Assess and Compare Community Taxonomic Structure
Individual Samples Compare Categories
16
Assess Alpha DiversityInter-sample Divesity
What does it tell you?
• Species count (observed species)
• Species richness (e.g., chao1)
• Species dominance (Fisher )• Diversity Index (Shannon)
• Phylogenetic diversity (PD_whole_tree)
Evaluate Sampling Depth (rarefaction plots)
17
Statistics– Alpha Diversitycompare_alpha_diversity.py
Beta Diversity StudiesInter‐Sample Diversity
Principal Coordinate Analysis Used
to Compare Microbiomes
2 Dimensional 3 Dimensional
19
Compare Groups (ADONIS*)(compare_categories.py)
*Analysis of variance using distance matrices
SOME EXAMPLES
20
SCIENCE VOL 332 ‐ 27 MAY 2011
Bacterial and Archaeal Taxa Associated with Disease Suppressiveness.
Shown are taxa that are more abundant in (i) suppressive (S) than in conducive (C) soil (pie A), (ii) “transplantation soil” (C+10%S) than in C (pie C), and (iii) S amended with R. solani (Sr) than in S (pie F). Pairwise comparisons (N = 4) depict the compositions of the top 10% of most dynamic taxa.
21
Fig 3. Distribution of fungal operational taxonomic units (OTUs) between different fungal phyla in the rhizosphere (A) and roots (B)
22
Figure 4. The top ten fungal operational taxonomic units (OTUs) (according to Similarity Percentage analysis) (SIMPER) contributing to the observed differences between control and Verticilliumdahliae treatments in Honeoye and Florence grown in conventionally and organically managed soils.
A) Rhizosphere soil (29–39%)B) B) Roots (61–74%).
doi:10.1371/journal.pone.0111455.g004
23
Shifts in Taxon Abundance and Co-occurrence Network as Effects of Warming
Chengwei Luo et al. Appl. Environ. Microbiol. 2014;80:1777-1786
Changes in relative abundance of pathways as an effect of warming
The heat map on the left represents changes in the abundance of different pathways (rows) for each pair of samples (columns), color coded based on the magnitude of the change (see scale on the top left). For selected pathways related to the emission of greenhouse gases, the relative abundances of the individual genes that constitute the pathways are shown on the right (small heat maps; rows represent samples, and columns represent genes).
25
Comparison of taxonomic
diversity (both bacteria and plants) and
catabolic diversity
CC = Cedar Creek KBS = Kellogg Biological Station
Comparison of Taxonomic Diversity (both bacteria and plants) and Catabolic Diversity
Phylogenetic Dissimilarities Metagenome Dissimilarities Catabolic Profiles
26
Relative Abundance of Bacterial Taxa – N2 GradientKellogg Biological Station (KBS)
Selected gene categories that changed in relative abundance across the N gradient
Kellogg Biological Station
27
Selected substrates for which the relative catabolic rates changes across the N gradients
KBS
Comparative MetagenomicsA Tool Bag for Rational Discovery
• Comparative metagenomics is the study of differences in the overall metabolism in a sample
– Measure the enzymes catalyzing metabolite interconversion
– Assign functions/roles to microbiome present
– Utilize grid computing to identify genes (GI)
– Analyze differences among microbial communities:• Visually using enzyme pathways (e.g., iPATH2)
• Statistically analyzing
– Enzyme diversity and uniqueness
– Pathway Enrichment
– Correlation with contributing taxa
28
AN EXAMPLE WITH DECAYING TROPICAL WOOD
Analytical Steps in Pilot Study
Wood DNA454 WGS
Sequencing
MG‐RAST
iPATH2
KOBAS
MicrobiomeAnalysis
QIIME
Primer 6
DB queries
CAZy
DiaGrid
29
Decaying Wood: Metabolic Subsystems
Amino Acids and Deriva ves 5%
Carbohydrates 6%
Cell Division and Cell Cycle 3%
Cell Wall and Capsule 4%
Clustering‐based subsystems
6%
Cofactors, Vitamins, Prosthe c Groups, Pigments
5%
DNA Metabolism 4%
Dormancy and Sporula on
1%
Fa y Acids, Lipids, and Isoprenoids
4%
Iron acquisi on and metabolism 3%
Membrane Transport 4%
Metabolism of Aroma c Compounds
3%
Miscellaneous 5%
Mo lity and Chemotaxis 3% Nitrogen Metabolism
2%
Nucleosides and Nucleo des 4%
Phages, Prophages, Transposable elements,
Plasmids 3%
Phosphorus Metabolism
3%
Photosynthesis 0%
Potassium metabolism
2%
Protein Metabolism 5%
RNA Metabolism 4%
Regula on and Cell signaling 3%
Respira on 4%
Secondary Metabolism 1%
Stress Response 4%
Sulfur Metabolism 3%
Virulence, Disease and Defense
4%
Rela ve Abundance
Principal Components Analysis1 – Functional Diversity
1Principal Components Analysis was conducted using the PCA function in the MG‐RAST software for Hierarchical Classification option
30
Lignocellulose – Active Enzymes
Carbohydrate Metabolizing Enzymes1
1Predicted CAZymes and CAZy Families for proteins by search using DOE’s BESC KnowledgeBase v1.1
31
Principal Taxa Associated with CAZyAbundance
CAZyFamily
Activities Principal Taxa
GH3 ‐glucosidase xylanase Acidobacteria Bacteroidetes
LO2 Lignin oxidase Aryl alcohol oxidase Acidobacteria Sordariaceae
GT2Glycosyltransferase_2_3
Response_reg|Glyco_tranf_2_3
Bacteroidetes Geodermatophilus
GH2 ‐galactosidase ‐mannosidase Bacteroidetes Bacteroidetes
GH13 ‐amylase pullulanase Eubacterium Penicillium
CE1 cinnamoyl esterase xylan esterase Solibacter Bradyrhizobium
GT35glycogen phosphorylase
starch phosphorylase
Acidobacteria Koribacter
INTERACTIVE PATHWAY EXPLORER2
Evaluating pathways and enzyme richness
32
Purine metabolism
Secondary bile acidbiosynthesis
metabolismPhenylalanine
Chloroalkane andcloroalkene degradation
isoleucine degradationValine, leucine and
metabolismGlycerolipid
GluconeogenesisGlycolysis /
tryptophan biosynthesisPhenylalanine , tyrosine and
metabolismPyruvate
Taurine andhypotaurinemetabolism
Arachidonic acidmetabolism
Butanoatemetabolism
Fatty acid elongationin mitochondria
biosynthesisClavulanic acid
by cytochrom P450Metabolism of xenobiotics
Selenocompoundmetabolism
biosynthesisInsect hormone
metabolismSphingolipid
metabolismMethane
[B] Phytochemicalcompounds
degradationNaphthalene
polyketide productsBiosynthesis of type II
isoleucine biosynthesisValine, leucine and
biosynthesisIsoflavonoid
Fructose andmannose metabolism
resistancebeta−Lactam
[B] Proteoglycans
Brassinosteroidbiosynthesis
Fatty acidmetabolism
Glutathionemetabolism
Tyrosine metabolism
Glycosphingolipidbiosynthesis −lact and neolacto series
metabolismCaffeine
Anthocyaninbiosynthesis
hydrocarbon degradationPolycyclic aromatic
pyridine alkaloid biosynthesisTropane, piperidine and
biosynthesisCarotenoid
C5−Brancheddibasic acidmetabolism
biosynthesisIndole alkaloid
metabolism
Porphyrin andchlorophyll
N−Glycanbiosynthesis
biosynthesiscephalosporinPenicillin and
globoseries
Glycosphingolipidbiosynthesis −
Inositol phosphatemetabolism
biosynthesisStreptomycin
metabolismbeta−Alanine
unit biosynthesisPolyketide sugar
Nitrogenmetabolism
DDT degradation
degradationBenzoate
Glycine, serine andthreonine metabolism
degradationGlycosaminoglycan
biosynthesisLysine
biosynthesisMonoterpenoid
degradationToluene
proline metabolismArginine and
metabolismdicarboxylateGlyoxylate and
biosynthesisNovobiocin
biosynthesisPeptidoglycan
metabolismRiboflavin
biosynthesisSteroid hormone
Sulfurmetabolism
by folateOne carbon pool
proteins[B] Lipids biosynthesis
Pentose and glucuronateinterconversions
Biotinmetabolism
other terpenoid−quinonebiosynthesis
Ubiquinone and
biosynthesisFatty acid
metabolismPentose phosphate
degradationXylene
Terpenoid backbonebiosynthesis
degradationBisphenol
D−glutamatemetabolism
D−Glutamine and
Steroid biosynthesis
Biosynthesis of type IIpolyketide backbone
Aminobenzoatedegradation
metabolismVitamin B6
Dioxin degradation
Biosynthesis ofunsaturated fatty acids
biosynthesisPuromycin
metabolismD−Alanine
metabolismLipoic acid
Cutin, suberine andwax biosynthesis
Nitrotoluenedegradation
[B] Glycosyltransferases
Butirosin andneomycin biosynthesis
ganglioseries
Glycosphingolipidbiosynthesis −
Glycosaminoglycanbiosynthesis −chondroitin sulfate
Pantothenate and CoAbiosynthesis
degradation ofKetone bodies
Synthesis and
metabolismThiamine
Ether lipidmetabolism
Flavonoidbiosynthesis
carboxylate cycleReductive
(CO2 fixation)
Glycosaminoglycan
heparan sulfatebiosynthesis −
metabolismCystein and methionine
glutamate metabolismAlanine , aspartate and
Other types ofO−glycan biosynthesis
Nicotinate andnicotinamidemetabolism
metabolism
Amino sugar andnucleotide sugar
biosynthesisZeatin
Ascorbate andaldarate metabolism
pinene degradationLimonene and
metabolismPropanoate
degradationCaprolactam
N−glycan biosynthesisVarious types of
Phenylpropanoidbiosynthesis
phosphorylationOxidative
Histidine metabolism
metabolismTryptophan
Citrate cycle(TCA cycle)
biosynthesisSesquiterpenoid
Other glycandegradation
Mucin typeO−glycan biosynthesis
ansamycinsBiosynthesis of
metabolismRetinol
metabolismGlycerophospholipid
biosynthesisPrimary bile acid
Photosynthesis −antenna proteins
D−ornithinemetabolism
D−Arginine and
biosynthesisGlucosinolate
biosynthesis
Flavone andflavonol
[B] Cytochrome P450
biosynthesisFolate
Photosynthesis
Pyrimidine metabolismmetabolismalpha−Linolenic acid
biosynthesis −keratan sulfate
Glycosaminoglycan
biosynthesisLipopolysaccharide
Galactosemetabolism
[B] Lipids
fixationCarbon
biosynthesisIsoquinoline alkaloid
nonribosomalpeptides
siderophore groupBiosynthesis of
Chlorocyclohexane andchlorobenzene degradation
Phosphonate andphosphinate metabolism
Tetracyclinebiosynthesis
degradationAtrazine
degradationStyrene
biosynthesisBenzoxazinone
metabolismStarch and sucrose
− other enzymesDrug metabolism
(GPI)−anchor biosynthesisGlycosylphosphatidylinositol
metabolismLinoleic acid
16−membered macrolidesBiosynthesis of 12−, 14− and
Ethylbenzenedegradation
proteins[B] Photosynthesis
Cyanoamino acidmetabolism
Biosynthesis of vancomycingroup antibiotics
− cytochrom P450Drug metabolism
degradationFluorobenzoate
Lysinedegradation
biosynthesisDiterpenoid
biosynthesisAcridone alkaloid
Metabolism ofTerpenoids and Polyketides
NucleotideMetabolism
and MetabolismGlycan Biosynthesis
Amino AcidMetabolism
Metabolism ofOther Amino Acid
and MetabolismXenobiotics Biodegradation
Other Secondary MetabolitesBiosynthesis of
MetabolismEnergy
CarbohydrateMetabolism
Cofactors and VitaminsMetabolism of
MetabolismLipid
Decaying wood meta‐metabolome: Metabolic Pathways
Purine metabolism
Secondary bile acidbiosynthesis
metabolismPhenylalanine
Chloroalkane andcloroalkene degradation
isoleucine degradationValine, leucine and
metabolismGlycerolipid
GluconeogenesisGlycolysis /
tryptophan biosynthesisPhenylalanine , tyrosine and
metabolismPyruvate
Taurine andhypotaurinemetabolism
Arachidonic acidmetabolism
Butanoatemetabolism
Fatty acid elongationin mitochondria
biosynthesisClavulanic acid
by cytochrom P450Metabolism of xenobiotics
Selenocompoundmetabolism
biosynthesisInsect hormone
metabolismSphingolipid
metabolismMethane
[B] Phytochemicalcompounds
degradationNaphthalene
polyketide productsBiosynthesis of type II
isoleucine biosynthesisValine, leucine and
biosynthesisIsoflavonoid
Fructose andmannose metabolism
resistancebeta−Lactam
[B] Proteoglycans
Brassinosteroidbiosynthesis
Fatty acidmetabolism
Glutathionemetabolism
Tyrosine metabolism
Glycosphingolipidbiosynthesis −lact and neolacto series
metabolismCaffeine
Anthocyaninbiosynthesis
hydrocarbon degradationPolycyclic aromatic
pyridine alkaloid biosynthesisTropane, piperidine and
biosynthesisCarotenoid
C5−Brancheddibasic acidmetabolism
biosynthesisIndole alkaloid
metabolism
Porphyrin andchlorophyll
N−Glycanbiosynthesis
biosynthesiscephalosporinPenicillin and
globoseries
Glycosphingolipidbiosynthesis −
Inositol phosphatemetabolism
biosynthesisStreptomycin
metabolismbeta−Alanine
unit biosynthesisPolyketide sugar
Nitrogenmetabolism
DDT degradation
degradationBenzoate
Glycine, serine andthreonine metabolism
degradationGlycosaminoglycan
biosynthesisLysine
biosynthesisMonoterpenoid
degradationToluene
proline metabolismArginine and
metabolismdicarboxylateGlyoxylate and
biosynthesisNovobiocin
biosynthesisPeptidoglycan
metabolismRiboflavin
biosynthesisSteroid hormone
Sulfurmetabolism
by folateOne carbon pool
proteins[B] Lipids biosynthesis
Pentose and glucuronateinterconversions
Biotinmetabolism
other terpenoid−quinonebiosynthesis
Ubiquinone and
biosynthesisFatty acid
metabolismPentose phosphate
degradationXylene
Terpenoid backbonebiosynthesis
degradationBisphenol
D−glutamatemetabolism
D−Glutamine and
Steroid biosynthesis
Biosynthesis of type IIpolyketide backbone
Aminobenzoatedegradation
metabolismVitamin B6
Dioxin degradation
Biosynthesis ofunsaturated fatty acids
biosynthesisPuromycin
metabolismD−Alanine
metabolismLipoic acid
Cutin, suberine andwax biosynthesis
Nitrotoluenedegradation
[B] Glycosyltransferases
Butirosin andneomycin biosynthesis
ganglioseries
Glycosphingolipidbiosynthesis −
Glycosaminoglycanbiosynthesis −chondroitin sulfate
Pantothenate and CoAbiosynthesis
degradation ofKetone bodies
Synthesis and
metabolismThiamine
Ether lipidmetabolism
Flavonoidbiosynthesis
carboxylate cycleReductive
(CO2 fixation)
Glycosaminoglycan
heparan sulfatebiosynthesis −
metabolismCystein and methionine
glutamate metabolismAlanine , aspartate and
Other types ofO−glycan biosynthesis
Nicotinate andnicotinamidemetabolism
metabolism
Amino sugar andnucleotide sugar
biosynthesisZeatin
Ascorbate andaldarate metabolism
pinene degradationLimonene and
metabolismPropanoate
degradationCaprolactam
N−glycan biosynthesisVarious types of
Phenylpropanoidbiosynthesis
phosphorylationOxidative
Histidine metabolism
metabolismTryptophan
Citrate cycle(TCA cycle)
biosynthesisSesquiterpenoid
Other glycandegradation
Mucin typeO−glycan biosynthesis
ansamycinsBiosynthesis of
metabolismRetinol
metabolismGlycerophospholipid
biosynthesisPrimary bile acid
Photosynthesis −antenna proteins
D−ornithinemetabolism
D−Arginine and
biosynthesisGlucosinolate
biosynthesis
Flavone andflavonol
[B] Cytochrome P450
biosynthesisFolate
Photosynthesis
Pyrimidine metabolismmetabolismalpha−Linolenic acid
biosynthesis −keratan sulfate
Glycosaminoglycan
biosynthesisLipopolysaccharide
Galactosemetabolism
[B] Lipids
fixationCarbon
biosynthesisIsoquinoline alkaloid
nonribosomalpeptides
siderophore groupBiosynthesis of
Chlorocyclohexane andchlorobenzene degradation
Phosphonate andphosphinate metabolism
Tetracyclinebiosynthesis
degradationAtrazine
degradationStyrene
biosynthesisBenzoxazinone
metabolismStarch and sucrose
− other enzymesDrug metabolism
(GPI)−anchor biosynthesisGlycosylphosphatidylinositol
metabolismLinoleic acid
16−membered macrolidesBiosynthesis of 12−, 14− and
Ethylbenzenedegradation
proteins[B] Photosynthesis
Cyanoamino acidmetabolism
Biosynthesis of vancomycingroup antibiotics
− cytochrom P450Drug metabolism
degradationFluorobenzoate
Lysinedegradation
biosynthesisDiterpenoid
biosynthesisAcridone alkaloid
Metabolism ofTerpenoids and Polyketides
NucleotideMetabolism
and MetabolismGlycan Biosynthesis
Amino AcidMetabolism
Metabolism ofOther Amino Acid
and MetabolismXenobiotics Biodegradation
Other Secondary MetabolitesBiosynthesis of
MetabolismEnergy
CarbohydrateMetabolism
Cofactors and VitaminsMetabolism of
MetabolismLipid
Tropical soil metametabolome: Metabolic Pathways
33
Purine metabolism
Secondary bile acidbiosynthesis
metabolismPhenylalanine
Chloroalkane andcloroalkene degradation
isoleucine degradationValine, leucine and
metabolismGlycerolipid
GluconeogenesisGlycolysis /
tryptophan biosynthesisPhenylalanine , tyrosine and
metabolismPyruvate
Taurine andhypotaurinemetabolism
Arachidonic acidmetabolism
Butanoatemetabolism
Fatty acid elongationin mitochondria
biosynthesisClavulanic acid
by cytochrom P450Metabolism of xenobiotics
Selenocompoundmetabolism
biosynthesisInsect hormone
metabolismSphingolipid
metabolismMethane
[B] Phytochemicalcompounds
degradationNaphthalene
polyketide productsBiosynthesis of type II
isoleucine biosynthesisValine, leucine and
biosynthesisIsoflavonoid
Fructose andmannose metabolism
resistancebeta−Lactam
[B] Proteoglycans
Brassinosteroidbiosynthesis
Fatty acidmetabolism
Glutathionemetabolism
Tyrosine metabolism
Glycosphingolipidbiosynthesis −lact and neolacto series
metabolismCaffeine
Anthocyaninbiosynthesis
hydrocarbon degradationPolycyclic aromatic
pyridine alkaloid biosynthesisTropane, piperidine and
biosynthesisCarotenoid
C5−Brancheddibasic acidmetabolism
biosynthesisIndole alkaloid
metabolism
Porphyrin andchlorophyll
N−Glycanbiosynthesis
biosynthesiscephalosporinPenicillin and
globoseries
Glycosphingolipidbiosynthesis −
Inositol phosphatemetabolism
biosynthesisStreptomycin
metabolismbeta−Alanine
unit biosynthesisPolyketide sugar
Nitrogenmetabolism
DDT degradation
degradationBenzoate
Glycine, serine andthreonine metabolism
degradationGlycosaminoglycan
biosynthesisLysine
biosynthesisMonoterpenoid
degradationToluene
proline metabolismArginine and
metabolismdicarboxylateGlyoxylate and
biosynthesisNovobiocin
biosynthesisPeptidoglycan
metabolismRiboflavin
biosynthesisSteroid hormone
Sulfurmetabolism
by folateOne carbon pool
proteins[B] Lipids biosynthesis
Pentose and glucuronateinterconversions
Biotinmetabolism
other terpenoid−quinonebiosynthesis
Ubiquinone and
biosynthesisFatty acid
metabolismPentose phosphate
degradationXylene
Terpenoid backbonebiosynthesis
degradationBisphenol
D−glutamatemetabolism
D−Glutamine and
Steroid biosynthesis
Biosynthesis of type IIpolyketide backbone
Aminobenzoatedegradation
metabolismVitamin B6
Dioxin degradation
Biosynthesis ofunsaturated fatty acids
biosynthesisPuromycin
metabolismD−Alanine
metabolismLipoic acid
Cutin, suberine andwax biosynthesis
Nitrotoluenedegradation
[B] Glycosyltransferases
Butirosin andneomycin biosynthesis
ganglioseries
Glycosphingolipidbiosynthesis −
Glycosaminoglycanbiosynthesis −chondroitin sulfate
Pantothenate and CoAbiosynthesis
degradation ofKetone bodies
Synthesis and
metabolismThiamine
Ether lipidmetabolism
Flavonoidbiosynthesis
carboxylate cycleReductive
(CO2 fixation)
Glycosaminoglycan
heparan sulfatebiosynthesis −
metabolismCystein and methionine
glutamate metabolismAlanine , aspartate and
Other types ofO−glycan biosynthesis
Nicotinate andnicotinamidemetabolism
metabolism
Amino sugar andnucleotide sugar
biosynthesisZeatin
Ascorbate andaldarate metabolism
pinene degradationLimonene and
metabolismPropanoate
degradationCaprolactam
N−glycan biosynthesisVarious types of
Phenylpropanoidbiosynthesis
phosphorylationOxidative
Histidine metabolism
metabolismTryptophan
Citrate cycle(TCA cycle)
biosynthesisSesquiterpenoid
Other glycandegradation
Mucin typeO−glycan biosynthesis
ansamycinsBiosynthesis of
metabolismRetinol
metabolismGlycerophospholipid
biosynthesisPrimary bile acid
Photosynthesis −antenna proteins
D−ornithinemetabolism
D−Arginine and
biosynthesisGlucosinolate
biosynthesis
Flavone andflavonol
[B] Cytochrome P450
biosynthesisFolate
Photosynthesis
Pyrimidine metabolismmetabolismalpha−Linolenic acid
biosynthesis −keratan sulfate
Glycosaminoglycan
biosynthesisLipopolysaccharide
Galactosemetabolism
[B] Lipids
fixationCarbon
biosynthesisIsoquinoline alkaloid
nonribosomalpeptides
siderophore groupBiosynthesis of
Chlorocyclohexane andchlorobenzene degradation
Phosphonate andphosphinate metabolism
Tetracyclinebiosynthesis
degradationAtrazine
degradationStyrene
biosynthesisBenzoxazinone
metabolismStarch and sucrose
− other enzymesDrug metabolism
(GPI)−anchor biosynthesisGlycosylphosphatidylinositol
metabolismLinoleic acid
16−membered macrolidesBiosynthesis of 12−, 14− and
Ethylbenzenedegradation
proteins[B] Photosynthesis
Cyanoamino acidmetabolism
Biosynthesis of vancomycingroup antibiotics
− cytochrom P450Drug metabolism
degradationFluorobenzoate
Lysinedegradation
biosynthesisDiterpenoid
biosynthesisAcridone alkaloid
Metabolism ofTerpenoids and Polyketides
NucleotideMetabolism
and MetabolismGlycan Biosynthesis
Amino AcidMetabolism
Metabolism ofOther Amino Acid
and MetabolismXenobiotics Biodegradation
Other Secondary MetabolitesBiosynthesis of
MetabolismEnergy
CarbohydrateMetabolism
Cofactors and VitaminsMetabolism of
MetabolismLipid
Decaying Wood Meta‐metabolome
Enzyme Enrichment of the EY Log Meta‐Metabolome
KO‐Entry
Mean Enrichment
KEGG Definition Role
K15726 24.39 cobalt‐zinc‐cadmium resistance protein CzcA Socio
K07344 8.20 type IV secretion system protein TrbL Conj
K03761 8.07 Major Facilitator Superfamily (MFS) transporter Transp
K04754 7.21 lipoprotein Memb
K07552 6.91DHA1 family, bicyclomycin/chloramphenicol resistance protein
Socio
K01690 6.25 phosphogluconate dehydratase [EC:4.2.1.12] Carb
K02487 6.13type IV pili sensor histidine kinase/response regulator
Socio
K06596 6.03 chemosensory pili system protein ChpA Socio
K00370 5.88 nitrate reductase 1, alpha subunit [EC:1.7.99.4] Metab
K11942 5.59 methylmalonyl‐CoA mutase [EC:5.4.99.2] Metab
K02067 5.55 ABC transport system substrate‐binding protein Transp
K15727 5.54 cobalt‐zinc‐cadmium resistance protein CzcB Socio
34
Conclusions
• Useful toolkit to analyze complex samples
• Evaluate microbial community structure and dynamic
• Cost‐effective DNA sequencing technology permits the analysis of a large number of samples
• Available, public domain, bioinformatics tools allows for detail and comprehensive analyses
• Has real and economic value in the analyses of soil characteristics, health and fertility
• Can be used effectively in soil management
Recommended