1. Extracting
genomes from
metagenomes
Mads Albertsen
Advanced Bacteriology @ KU
27-09-2013
CENTER FOR MICROBIAL COMMUNITIES

2. Agenda
Why do we need genomes?
How can we get them?
… and then what?
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Slides:

3. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Who - when, where and why?

4. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Water and wastewater treatment
Diseases and infectionsEnergy
Local and global challenges

5. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Genome = Parts list with 3000-5000 items
What is a genome?

6. 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

7. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
How do we get the genomes?
What you think you study What you actually study

8. Single cell genomics
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
How do we get the genomes?
Culturing
Few microorganisms can be easily cultured (<<5%)
Microorganisms needs to be studied in their environment
Only routinely performed in specialized labs
Very incomplete genomes (mean 40%, range 10-90%)
https://www.bigelow.org/

9. Single cell genomics
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
How do we get the genomes?
Culturing
Few microorganisms can be easily cultured (<<5%)
Microorganisms needs to be studied in their environment
Only routinely performed in specialized labs
Very incomplete genomes (mean 40%, range 10-90%)
Metagenomics
https://www.bigelow.org/

10. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Genome = Parts list of a single species
What is a genome?

11. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Metagenome = Parts list of the community
Photo: D. Kunkel; color, E. Latypova
What is a metagenome?

12. ”...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?

13. 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!

14. 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

15. 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)

16. DNA extraction
Sequencing
Assembly
Contigs Search against
database
Phylogenetic classification
Who is there?
Functional classification
What can they do?
Bacterium A
Bacterium B
...
Bacterium X
Gene A
Gene B
...
Gene X
100++ Abundant
species (≈3 Mbp each)
1000+ bp
100-150 bp
Reads
Metagenomics
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

17. Metagenomics
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
”If you want to
understand the ecosystem
you need to
understand the individual species
in the ecosystem”

18. Metagenomics
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Lion + Eagle ≠ Flying Lion

19. 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)

20. 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)

21. 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

22. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Low micro-diversityHigh micro-diversity
Short term
enrichment
Extracting genomes

23. 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)

24. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Genomic signatures:
- GC / Codon usage
- Tetranucleotide frequency + statistical method
Complex sample
PhD student
”Binning”
Binning

25. 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

26. Sequence composition-independent binning
Sample 1
Abundance
Sample 2
Abundance
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Binning

27. Sequence composition-independent binning
Sample 1 Sample 2
Abundance Sample 1
AbundanceSample2
Abundance
Abundance
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Binning

28. 1. Reduce micro-diversity
2. Use multiple related samples
Abundance Sample 1
AbundanceSample2
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Binning

29. 1. Reduce micro-diversity
2. Use multiple related samples
Abundance Sample 1
AbundanceSample2
Abundance Sample 1
AbundanceSample2
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Binning

30. 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

31. Short term
enrichment
Full-scale EBPR plant
SBR reactor
Days 1. Reduction of (micro)-diversity
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITYAlbertsen et al., 2013 Nat. Biotech.

32. Short term
enrichment
Full-scale EBPR plant
SBR reactor
2. Two
different
DNA
extraction
methods
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITYAlbertsen et al., 2013 Nat. Biotech.

33. Colored using a set of 100 phylogenetic marker genes
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITYAlbertsen et al., 2013 Nat. Biotech.

34. 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.

35. 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.

36. 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.

37. 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.

38. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITYAlbertsen et al., 2013 Nat. Biotech.
Phyla
Genes (HMM models)
Essential single copy genesAssembly inspection
Genome validation

39. 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

40. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
...add more samples!
Complex samples
S. M. Karst, AAU

41. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
It’s just a potential!
..and a poorly translated description of it.

42. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Metabolites
Proteins
mRNA
DNA
Meta-bolomics
Meta-proteomics
Meta-transcriptomics
Meta-genomics
In Situ methods
Community structure Microbial functions
Extraction
P-Removal:
N-Removal:
-Removal:
Foaming:
Ethanol production:
Microbial needs
Understanding ecosystems

43. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Understanding ecosystems
McIlroy and Albertsen et al., 2013 Candidatus Competibacter’-lineage genomes
retrieved 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

44. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Understanding ecosystems
McIlroy and Albertsen et al., 2013 Candidatus Competibacter’-lineage genomes
retrieved from metagenomes reveal functional metabolic diversity. ISME J (in press).
FISH with Competibacter
specific probe
MAR with H3-labeled glucose

45. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Understanding ecosystems
Genomes enable comprehensive transcriptomics of individual
species in complex communities.
(stranded mRNAseq data)

46. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
G.W. Tyson
Per H. Nielsen
Simon J. McIllroy
Søren M. Karst
EB group
C. Dorringer H. Daims M. Wagner
P. Hugenholtz
University of Vienna
University of Queensland
Questions? @MadsAlbertsen85
MadsAlbertsen
ma@bio.aau.dk