[2013.09.27] extracting genomes from metagenomes
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Extracting genomes from metagenomes
Mads AlbertsenAdvanced Bacteriology @ KU
27-09-2013
CENTER FOR MICROBIAL COMMUNITIES
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
AAAAAAAAAAAAAA
AAAAAAAAATAAAA
AAAAAAAAACAAAA
AAAAAAAAA
TAAAA
CAAAA
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
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
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
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
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
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
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
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
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
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
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Understanding ecosystems
Genomes enable comprehensive transcriptomics of individual species in complex communities.
(stranded mRNAseq data)
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
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
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