Extracting genomes from metagenomes

Mads AlbertsenPhD Student (2011-2014)

02-12-2013 @ University of Vienna

CENTER FOR MICROBIAL COMMUNITIES

Aalborg

Per H. Nielsen

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

Microbial Ecology: Who - when, where and why?

1/13

Seweragesystem

Occasional breakdowns

Strike

Microbial Ecology

Nielsen et al., 2012 Curr. Opin. Biotechnol. 23:452-9 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

Biological wastewater treatment

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

Aalborg

Hjørring

Århus

Odense

MiDASSince 2006 4 samples / year = 7 2 samples / year = 6 Some years = 16

Copenhagen

Nielsen et al., 2012 Curr. Opin. Biotechnol. 23:452-9

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

30 abundant core genera in all Danish

EBPR WWTPs

Functional studies using MAR-FISH

Nielsen et al., 2012 Curr. Opin. Biotechnol. 23:452-9

qFISH

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www.midasfieldguide.org

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

Metabolites

Proteins

mRNA

DNA

Meta-bolomics

Meta-proteomics

Meta-transcriptomics

Meta-genomics

Data integration

In Situ methods

Community structure Microbial functions

Omics

P-Removal:

N-Removal:

-Removal:

Foaming:

Ethanol production:

Microbial needsEcology

Understanding ecosystems

Albertsen et al., 2012, ISME J 6: 1094-106

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

Metabolites

Proteins

mRNA

DNA

Meta-bolomics

Meta-proteomics

Meta-transcriptomics

Meta-genomics

Data integration

In Situ methods

Community structure Microbial functions

Omics

P-Removal:

N-Removal:

-Removal:

Foaming:

Ethanol production:

Microbial needsEcology

Understanding ecosystems

Omics requires good reference genomes!

Albertsen et al., 2012, ISME J 6: 1094-106

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITYAlbertsen et al., 2012, ISME J 6: 1094-106

Available genomes (+)

(+)

(+)

Culturing

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

How do we get the genomes?

Few microorganisms can be easily cultured (<<5%)

Tetrasphaera: Kristiansen et al., 2013, ISME J 7: 543-54Microthirx: McIllroy et al., 2013, ISME J 7:1161-72

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How do we get the genomes?

What you think you study What you actually study

Single cell genomics

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How do we get the genomes?

CulturingFew microorganisms can be easily cultured (<<5%)

Only routinely performed in specialized labsVery incomplete genomes (mean 40%, range 10-90%)

www.bigelow.org

Single cell genomics

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How do we get the genomes?

CulturingFew microorganisms can be easily cultured (<<5%)

Only routinely performed in specialized labsVery incomplete genomes (mean 40%, range 10-90%)

Metagenomics

www.bigelow.org

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Genome = Parts list of a single species

What is a genome?

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Metagenome = Parts list of the community

Photo: D. Kunkel; color, E. Latypova

What is a metagenome?

”...functional analysis of the collective genomes of soil microflora, which we term the metagenome of the soil.”

- J. Handelsman et al., 1998

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What is a metagenome?

PubMed: metagenom*[Title/Abstract]

”...functional analysis of the collective genomes of soil microflora, which we term the metagenome of the soil.”

- J. Handelsman et al., 1998

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Metagenomics is hot!

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”...functional analysis of the collective genomes of soil microflora, which we term the metagenome of the soil.”

- J. Handelsman et al., 1998

PubMed: metagenom*[Title/Abstract]

Sequencing costs

http://www.genome.gov/sequencingcosts/

Sequencing is cheap!

DNA extraction

Sequencing

Assembly Contigs Search against

database

1000+ bp

100-150 bp

Reads

Metagenomics

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

100++ Abundant species (≈3 Mbp each)

DNA extraction

Sequencing

Assembly Contigs Search against

database

Phylogenetic classificationWho is there?

Functional classificationWhat can they do?

Bacterium ABacterium B...Bacterium X

Gene AGene B...Gene X

100++ Abundant species (≈3 Mbp each)

1000+ bp

100-150 bp

Reads

Metagenomics

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

DNA extraction

Sequencing

Assembly Contigs Search against

database

Phylogenetic classificationWho is there?

Functional classificationWhat can they do?

Bacterium ABacterium B...Bacterium X

Gene AGene B...Gene X

100++ Abundant species (≈3 Mbp each)

1000+ bp

100-150 bp

Reads

Metagenomics

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Omics requires good reference genomes!

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”If you want to understand the ecosystem

you need to understand the individual species

in the ecosystem”

Metagenomics

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Lion + Eagle ≠ Flying Lion

DNA extraction

Sequencing

Assembly

100-150 bp

Reads

Metagenomics

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

Why not full genomes?

100++ Abundant species (≈3 Mbp each)

Contigs

1000+ bp

DNA extraction

Sequencing

Assembly Contigs

1000+ bp

100-150 bp

Reads

Metagenomics

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Why not full genomes?

1. Micro-diversity

2. Separation of genomes (Binning)

100++ Abundant species (≈3 Mbp each)

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Not 1 strain

Many closely related strains

AAAAAAAAAAAAAA

AAAAAAAAATAAAA

AAAAAAAAACAAAA

AAAAAAAAA

TAAAA

CAAAA

What you get

AAAAA

Assembly

Micro-diversity

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Low micro-diversityHigh micro-diversity

Short term enrichment

Micro-diversity

DNA extraction

Sequencing

Assembly

100-150 bp

Reads

Binning

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

Why not full genomes?

1. Micro-diversity

2. Separation of genomes (Binning)

100++ Abundant species (≈3 Mbp each)

Contigs

1000+ bp

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Complex sample

PhD student

”Binning”

Binning

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Genomic signatures (e.g GC and codon usage )Tetranucleotide frequency + statistical method

Complex sample

PhD student

”Binning”

Binning

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Complex sample

PhD student

”Binning”

Short pieces of DNA sequences (1-10kbp)Local sequence divergence

BinningGenomic signatures (e.g GC and codon usage )Tetranucleotide frequency + statistical method

”Metagenomics can be used to measure the abundance of the

organims in the original sample.”

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CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

Binning

Assembly

ScaffoldsMetagenome reads Abundance

Sequencing

Original sample

Mapping 3x1x1x

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Binning

Assembly

ScaffoldsMetagenome reads Abundance

Sequencing

Original sample

Mapping 3x1x1x

Sample 1

Abun

danc

e

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Binning

Sequence composition-independent binning

Sample 1

Abun

danc

e

Sample 2

Abun

danc

e

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Binning

Sequence composition-independent binning

Sequence composition-independent binning

Sample 1 Sample 2

Abundance Sample 1

Abun

danc

e Sa

mpl

e 2

Abun

danc

e

Abun

danc

e

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Binning

1. Reduce micro-diversity

2. Use multiple related samples

Abundance Sample 1

Abun

danc

e Sa

mpl

e 2

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Binning

1. Reduce micro-diversity

2. Use multiple related samples

Abundance Sample 1

Abun

danc

e Sa

mpl

e 2

Abundance Sample 1

Abun

danc

e Sa

mpl

e 2

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

Binning

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITYH. Daims & C. Dorninger, DOME, University of Vienna

• Nitrospira enrichment running for years

• 3 dominant species

• No micro-diversity

Binning

Short term enrichment

Full-scale EBPR plantSBR reactor

Days 1. Reduction of (micro)-diversityCENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITYAlbertsen et al., 2013 Nat. Biotech.

Short term enrichment

Full-scale EBPR plantSBR reactor

2. Two different

DNA extraction methods

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITYAlbertsen et al., 2013 Nat. Biotech.

Colored using a set of 100 phylogenetic marker genes

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITYAlbertsen et al., 2013 Nat. Biotech.

Colored using a set of 100 phylogenetic marker genes

TM7-1 (1.6%)

TM7-2 (0.7%)

TM7-3 (0.2%)

TM7-4 (0.06%)

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITYAlbertsen et al., 2013 Nat. Biotech.

Zoom on target

TM7-2 (0.7%)

Colored using a set of 100 phylogenetic marker genes

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITYAlbertsen et al., 2013 Nat. Biotech.

Zoom on target

PC2

PC1

TM7-2

PCA on genomic signatures

TM7-2 (0.7%)

Colored using a set of 100 phylogenetic marker genes

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITYAlbertsen et al., 2013 Nat. Biotech.

Colored using a set of 100 phylogenetic marker genes

TM7-1 (1.6%)

Candidate phylum TM7

Saccharibacteria

Candidatus Saccharimonas aalborgensis

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITYAlbertsen et al., 2013 Nat. Biotech.

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITYAlbertsen et al., 2013 Nat. Biotech.

Phyla

Genes (HMM models)

Essential single copy genesAssembly inspection

Genome validation

In situ confirmation

PL. Larsen, SJ. McIllroy

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITYAlbertsen et al., 2013 Nat. Biotech.

http://madsalbertsen.github.io/multi-metagenome/Short: goo.gl/0ctA3

• Guides• Workflow scripts• Example data• All the code• Reccomendations

R markdown enables reproducible and

transparent genome extractions

Multi-metagenome

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It’s just a potential!

..and a poor description of it.

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Competibacter

McIlroy and Albertsen et al., 2013, ISME J (AOP).

Competibacter has the potential to negatively influence phosphorus removal in wastewater treatment.

Litterature disagreement on glycolytic pathways with consequences for modeling.

Candidatus Competibacter odensis

(44%)

GAO989

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Competibacter

FISH with Competibacter specific probe

MAR with H3-labeled glucose

McIlroy and Albertsen et al., 2013, ISME J (AOP).

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Obtaining genomes is easy…

… but they are useless without high quality annotations, in situ validations

and good questions!

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G.W. Tyson

Per H. NielsenSimon J. McIllroySøren M. KarstEB group

C. Dorringer H. Daims P. HugenholtzUniversity of Vienna

University of Queensland

Questions? @MadsAlbertsen85

MadsAlbertsenma@bio.aau.dk

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Databases

Contigs

Databases

...you only see what is in the database

Annotated metagenome

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What is in the databases?

PhylaClassOrderSpecies

2946

1001268

90249405

99322

Genomes 16S

Finshed Genomes in IMGVs.

Greengenes 16S rRNA database

Note: only including 1 strain pr. species

*97% clustering

*

MG-RAST example

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Contigs

650.000 EBPR proteins with taxonomy assigned

How similar are they to the genomes in the database?

Sludge microbes vs. Database genomes

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650.000 EBPR proteins

Note: not abundance weighted

Sludge microbes vs. Database genomes

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650.000 EBPR proteins1.260.000 Human gut

Qin et al., 2010 NatureRAST ID: 4448044.3

Note: not abundance weighted

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Sludge microbes vs. Database genomes

The 7 genera with most EBPR proteins assigned

Effect of missing genomes

What is the effect of not having closely related genomes in the database?

1. Remove a genome from the database

2. Search the removed genome against the database

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CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

Effect of missing genomes

Best hit

Bacteria 1268Proteobacteria 564Betaproteobacteria 84Rhodocyclales 5Rhodocyclaceae 5

Accumulibacter phosphatis

blastp

Related genomes

4326 proteins

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Effect of missing genomes

Best hit

Accumulibacter phosphatis

blastp

Related genomes

4326 proteinsAzoarcus

Bacteria 1268Proteobacteria 564Betaproteobacteria 84Rhodocyclales 5Rhodocyclaceae 5

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Effect of missing genomes

MEGAN LCA

Accumulibacter phosphatis

blastp

Lowest common ancester (LCA) approach:Hit 1: Beta-proteobacteria 80% IDHit 2: Gamma-proteobacteria 79% IDHit 3: Actinobacteria 59% ID

Assigned to Proteobacteria

Related genomes

4326 proteins

Bacteria 1268Proteobacteria 564Betaproteobacteria 84Rhodocyclales 5Rhodocyclaceae 5

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

Effect of missing genomes

MEGAN LCA

Accumulibacter phosphatis

blastp

Genus

No hits 261

Bacteria 325

Proteobacteria 860

Beta- 853

Rhodocyclaceae 1149

4326 proteins:• 27% correctly

classified on genus level

• 54% not assigned the correct class

• 101 genera identified

Related genomes

Lowest common ancester (LCA) approach:Hit 1: Beta-proteobacteria 80% IDHit 2: Gamma-proteobacteria 79% IDHit 3: Actinobacteria 59% ID

Assigned to Proteobacteria

4326 proteins

Bacteria 1268Proteobacteria 564Betaproteobacteria 84Rhodocyclales 5Rhodocyclaceae 5

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Effect of missing genomes

MEGAN LCA

Nitrospira defluvii

Bacteria 1268Nitrospirae 3

blastp

Related genomes

4268 proteins:• 1% correctly

classified on phylum level

Phylum

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Effect of missing genomes

MEGAN LCA+

KEGG

Nitrospira defluvii

blastp

Related genomesBacteria 1268Nitrospirae 3

What about function?

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Effect of missing genomes

MEGAN LCA+

KEGG

Nitrospira defluvii

blastp

Related genomesBacteria 1268Nitrospirae 3

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Effect of missing genomes

Nitrospira defluvii

blastp

Related genomes

MEGAN LCA+

KEGG

Bacteria 1268Nitrospirae 3

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Implication of missing genomes

Function A

Function B

Function C

Function D

Pitfalls

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You always get billions of data!