Extracting genomes from metagenomes

Mads AlbertsenAdvanced Bacteriology @ KU

27-09-2013

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Agenda

Why do we need genomes?

How can we get them?

… and then what?

CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITYSlides:

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Who - when, where and why?

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Water and wastewater treatment

Diseases and infectionsEnergy

Local and global challenges

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Genome = Parts list with 3000-5000 items

What is a genome?

Culturing

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How do we get the genomes?

Few microorganisms can be easily cultured (<<5%)Microorganisms needs to be studied in their environment

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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%)Microorganisms needs to be studied in their environment

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

https://www.bigelow.org/

Single cell genomics

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How do we get the genomes?

CulturingFew microorganisms can be easily cultured (<<5%)Microorganisms needs to be studied in their environment

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

Metagenomics

https://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 sexy!

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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

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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

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Metagenomics

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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 Contigs

1000+ bp

100-150 bp

Reads

Metagenomics

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Why not full genomes?

100++ Abundant species (≈3 Mbp each)

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

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What you get

AAAAA

Assembly

Extracting genomes

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Low micro-diversityHigh micro-diversity

Short term enrichment

Extracting genomes

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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Genomic signatures:- GC / Codon usage- Tetranucleotide frequency + statistical method

Complex sample

PhD student

”Binning”

Binning

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Genomic signatures:- GC / Codon usage- Tetranucleotide frequency + statistical method

Complex sample

PhD student

”Binning”

Problems:- Short pieces of sequence (1-10kbp)- Local sequence divergence

Binning

Sequence composition-independent binning

Sample 1

Abun

danc

e

Sample 2

Abun

danc

e

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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

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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

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

Multi-metagenome

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...add more samples!

Complex samples

S. M. Karst, AAU

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It’s just a potential!

..and a poorly translated description of it.

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Metabolites

Proteins

mRNA

DNA

Meta-bolomics

Meta-proteomics

Meta-transcriptomics

Meta-genomics

Data integration

In Situ methods

Community structure Microbial functions

Extraction

P-Removal:

N-Removal:

-Removal:

Foaming:

Ethanol production:

Microbial needsEcology

Understanding ecosystems

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Understanding ecosystems

McIlroy and Albertsen et al., 2013 Candidatus Competibacter’-lineage genomesretrieved from metagenomes reveal functional metabolic diversity. ISME J (in press).

• Competibacter has a potential to negatively effect phosphorus removal in wastewater treatment

• 2 Genomes obtained from enrichment metagenomes

• Compared to full-scale metagenomes• Only 1 abundant

• Genomic reconstruction reveals potential for fermentation of glucose

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Understanding ecosystems

McIlroy and Albertsen et al., 2013 Candidatus Competibacter’-lineage genomesretrieved from metagenomes reveal functional metabolic diversity. ISME J (in press).

FISH with Competibacter specific probe

MAR with H3-labeled glucose

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Understanding ecosystems

Genomes enable comprehensive transcriptomics of individual species in complex communities.

(stranded mRNAseq data)

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G.W. Tyson

Per H. NielsenSimon J. McIllroySøren M. KarstEB group

C. Dorringer H. Daims M. WagnerP. Hugenholtz

University of Vienna

University of Queensland

Questions? @MadsAlbertsen85

MadsAlbertsenma@bio.aau.dk