metagenomics: principles and applications - … nutrient workshop/2015...metagenomics: principles...

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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

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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

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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.

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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)

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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

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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

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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

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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

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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

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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

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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

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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

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• 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

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MOVING FROM RAW SEQUENCES TO USEFUL RESULTS

Workflow with QIIME

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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

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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)

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Statistics– Alpha Diversitycompare_alpha_diversity.py

Beta Diversity StudiesInter‐Sample Diversity

Principal Coordinate Analysis Used

to Compare Microbiomes

2 Dimensional 3 Dimensional

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Beta Diversity Statistics (group_significance.py)

Beta Diversity with Taxonomy

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Compare Groups (ADONIS*)(compare_categories.py)

*Analysis of variance using distance matrices

SOME EXAMPLES

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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.

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Fig 3. Distribution of fungal operational taxonomic units (OTUs) between different fungal phyla in the rhizosphere (A) and roots (B)

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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

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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).

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ASSESSING FUNCTIONAL DIVERSITY

The Meta‐metabolome

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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

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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

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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

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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

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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

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Lignocellulose – Active Enzymes

Carbohydrate Metabolizing Enzymes1

1Predicted CAZymes and CAZy Families for proteins by search using DOE’s BESC KnowledgeBase v1.1

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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

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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


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








metabolismPyruvate



Butanoatemetabolism







metabolismMethane








[B] Proteoglycans




Tyrosine metabolism


metabolismCaffeine







metabolism




globoseries






Nitrogenmetabolism

DDT degradation

degradationBenzoate



biosynthesisLysine


degradationToluene







Sulfurmetabolism




Biotinmetabolism


Ubiquinone and



degradationXylene




D−Glutamine and





Dioxin degradation









ganglioseries





Synthesis and

metabolismThiamine




(CO2 fixation)

Glycosaminoglycan






metabolism


biosynthesisZeatin















metabolismRetinol





D−Arginine and


biosynthesis

Flavone andflavonol

[B] Cytochrome P450

biosynthesisFolate

Photosynthesis



Glycosaminoglycan


Galactosemetabolism

[B] Lipids

fixationCarbon







degradationAtrazine

degradationStyrene













Lysinedegradation










MetabolismEnergy



MetabolismLipid

Tropical soil metametabolome: Metabolic Pathways

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Purine metabolism








metabolismPyruvate



Butanoatemetabolism







metabolismMethane








[B] Proteoglycans




Tyrosine metabolism


metabolismCaffeine







metabolism




globoseries






Nitrogenmetabolism

DDT degradation

degradationBenzoate



biosynthesisLysine


degradationToluene







Sulfurmetabolism




Biotinmetabolism


Ubiquinone and



degradationXylene




D−Glutamine and





Dioxin degradation









ganglioseries





Synthesis and

metabolismThiamine




(CO2 fixation)

Glycosaminoglycan






metabolism


biosynthesisZeatin















metabolismRetinol





D−Arginine and


biosynthesis

Flavone andflavonol

[B] Cytochrome P450

biosynthesisFolate

Photosynthesis



Glycosaminoglycan


Galactosemetabolism

[B] Lipids

fixationCarbon







degradationAtrazine

degradationStyrene













Lysinedegradation










MetabolismEnergy



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

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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

metagenomics: principles and applications - … nutrient workshop/2015...metagenomics: principles...

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