[2013.09.27] extracting genomes from metagenomes
TRANSCRIPT
![Page 1: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/1.jpg)
Extracting genomes from metagenomes
Mads AlbertsenAdvanced Bacteriology @ KU
27-09-2013
CENTER FOR MICROBIAL COMMUNITIES
![Page 2: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/2.jpg)
Agenda
Why do we need genomes?
How can we get them?
… and then what?
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITYSlides:
![Page 3: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/3.jpg)
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Who - when, where and why?
![Page 4: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/4.jpg)
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Water and wastewater treatment
Diseases and infectionsEnergy
Local and global challenges
![Page 5: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/5.jpg)
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Genome = Parts list with 3000-5000 items
What is a genome?
![Page 6: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/6.jpg)
Culturing
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
How do we get the genomes?
Few microorganisms can be easily cultured (<<5%)Microorganisms needs to be studied in their environment
![Page 7: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/7.jpg)
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
How do we get the genomes?
What you think you study What you actually study
![Page 8: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/8.jpg)
Single cell genomics
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
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/
![Page 9: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/9.jpg)
Single cell genomics
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
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/
![Page 10: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/10.jpg)
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Genome = Parts list of a single species
What is a genome?
![Page 11: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/11.jpg)
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Metagenome = Parts list of the community
Photo: D. Kunkel; color, E. Latypova
What is a metagenome?
![Page 12: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/12.jpg)
”...functional analysis of the collective genomes of soil microflora, which we term the metagenome of the soil.”
- J. Handelsman et al., 1998
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
What is a metagenome?
![Page 13: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/13.jpg)
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
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Metagenomics is sexy!
![Page 14: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/14.jpg)
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
”...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
![Page 15: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/15.jpg)
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)
![Page 16: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/16.jpg)
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
![Page 17: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/17.jpg)
Metagenomics
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
”If you want to understand the ecosystem
you need to understand the individual species
in the ecosystem”
![Page 18: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/18.jpg)
Metagenomics
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Lion + Eagle ≠ Flying Lion
![Page 19: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/19.jpg)
DNA extraction
Sequencing
Assembly Contigs
1000+ bp
100-150 bp
Reads
Metagenomics
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Why not full genomes?
100++ Abundant species (≈3 Mbp each)
![Page 20: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/20.jpg)
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)
![Page 21: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/21.jpg)
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Not 1 strain
Many closely related strains
AAAAAAAAAAAAAA
AAAAAAAAATAAAA
AAAAAAAAACAAAA
AAAAAAAAA
TAAAA
CAAAA
What you get
AAAAA
Assembly
Extracting genomes
![Page 22: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/22.jpg)
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Low micro-diversityHigh micro-diversity
Short term enrichment
Extracting genomes
![Page 23: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/23.jpg)
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)
![Page 24: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/24.jpg)
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Genomic signatures:- GC / Codon usage- Tetranucleotide frequency + statistical method
Complex sample
PhD student
”Binning”
Binning
![Page 25: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/25.jpg)
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
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
![Page 26: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/26.jpg)
Sequence composition-independent binning
Sample 1
Abun
danc
e
Sample 2
Abun
danc
e
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Binning
![Page 27: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/27.jpg)
Sequence composition-independent binning
Sample 1 Sample 2
Abundance Sample 1
Abun
danc
e Sa
mpl
e 2
Abun
danc
e
Abun
danc
e
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Binning
![Page 28: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/28.jpg)
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
![Page 29: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/29.jpg)
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
![Page 30: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/30.jpg)
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
![Page 31: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/31.jpg)
Short term enrichment
Full-scale EBPR plantSBR reactor
Days 1. Reduction of (micro)-diversityCENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITYAlbertsen et al., 2013 Nat. Biotech.
![Page 32: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/32.jpg)
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.
![Page 33: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/33.jpg)
Colored using a set of 100 phylogenetic marker genes
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITYAlbertsen et al., 2013 Nat. Biotech.
![Page 34: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/34.jpg)
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.
![Page 35: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/35.jpg)
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.
![Page 36: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/36.jpg)
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.
![Page 37: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/37.jpg)
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.
![Page 38: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/38.jpg)
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITYAlbertsen et al., 2013 Nat. Biotech.
Phyla
Genes (HMM models)
Essential single copy genesAssembly inspection
Genome validation
![Page 39: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/39.jpg)
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
![Page 40: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/40.jpg)
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
...add more samples!
Complex samples
S. M. Karst, AAU
![Page 41: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/41.jpg)
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
It’s just a potential!
..and a poorly translated description of it.
![Page 42: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/42.jpg)
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
Extraction
P-Removal:
N-Removal:
-Removal:
Foaming:
Ethanol production:
Microbial needsEcology
Understanding ecosystems
![Page 43: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/43.jpg)
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).
• 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
![Page 44: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/44.jpg)
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
![Page 45: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/45.jpg)
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Understanding ecosystems
Genomes enable comprehensive transcriptomics of individual species in complex communities.
(stranded mRNAseq data)
![Page 46: [2013.09.27] extracting genomes from metagenomes](https://reader038.vdocument.in/reader038/viewer/2022110118/554f4775b4c905524c8b46f1/html5/thumbnails/46.jpg)
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