[2013.09.27] extracting genomes from metagenomes

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  • [2013.09.27] extracting genomes from metagenomes

    1. 1. Extracting genomes from metagenomes Mads Albertsen Advanced Bacteriology @ KU 27-09-2013 CENTER FOR MICROBIAL COMMUNITIES
    2. 2. Agenda Why do we need genomes? How can we get them? … and then what? CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Slides:
    3. 3. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Who - when, where and why?
    4. 4. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Water and wastewater treatment Diseases and infectionsEnergy Local and global challenges
    5. 5. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Genome = Parts list with 3000-5000 items What is a genome?
    6. 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. 7. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY How do we get the genomes? What you think you study What you actually study
    8. 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. 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. 10. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Genome = Parts list of a single species What is a genome?
    11. 11. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Metagenome = Parts list of the community Photo: D. Kunkel; color, E. Latypova What is a metagenome?
    12. 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. 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. 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. 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. 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. 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. 18. Metagenomics CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Lion + Eagle ≠ Flying Lion
    19. 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. 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. 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. 22. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Low micro-diversityHigh micro-diversity Short term enrichment Extracting genomes
    23. 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. 24. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Genomic signatures: - GC / Codon usage - Tetranucleotide frequency + statistical method Complex sample PhD student ”Binning” Binning
    25. 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. 26. Sequence composition-independent binning Sample 1 Abundance Sample 2 Abundance CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Binning
    27. 27. Sequence composition-independent binning Sample 1 Sample 2 Abundance Sample 1 AbundanceSample2 Abundance Abundance CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Binning
    28. 28. 1. Reduce micro-diversity 2. Use multiple related samples Abundance Sample 1 AbundanceSample2 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Binning
    29. 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. 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. 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. 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. 33. Colored using a set of 100 phylogenetic marker genes CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITYAlbertsen et al., 2013 Nat. Biotech.
    34. 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. 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. 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. 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. 38. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITYAlbertsen et al., 2013 Nat. Biotech. Phyla Genes (HMM models) Essential single copy genesAssembly inspection Genome validation
    39. 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. 40. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY ...add more samples! Complex samples S. M. Karst, AAU
    41. 41. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY It’s just a potential! ..and a poorly translated description of it.
    42. 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. 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. 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. 45. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Understanding ecosystems Genomes enable comprehensive transcriptomics of individual species in complex communities. (stranded mRNAseq data)
    46. 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

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