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Assembly of metagenomes

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A talk for I gave for the 2011 metagenomics course at the Biological Dept. Univ. of Oslo April 2011

A talk for I gave for the 2011 metagenomics course at the Biological Dept. Univ. of Oslo April 2011

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Transcript

  • 1. Assembly of metagenomes
    Lex Nederbragt
    Norwegian Sequencing Center &
    Centre for Ecological and Evolutionary Synthesis
    University of Oslo
  • 2. What is assembly
    From reads to genome
  • 3. Why assembly?
    Wooley JC et al, PLoSComput Biol. 2010 Feb 26;6(2):e1000667
  • 4. How
    Find overlap between reads
  • 5. How
    Build consensus sequence
  • 6. Challenges
    Repetitive element
    DNA
    Shotgun
    reads
    Shotgun reads
    Contigs
    Collapsed contig
  • 7. Results
    Lots of pieces
  • 8. Mate pairs
  • 9. Assembly with mate pairs
    Gaps
    Paired reads
    Contigs
    Scaffold
  • 10. Mate pairs
    Contig
    Contig
    Contig
    Scaffold
    NNNNN
    NNNNN
  • 11. Mate pairs?
    454/Illumina
    Illumina
    150– 600 bases
  • 12. Mate pairs!
    Longer jumps:
  • 13. Mate pairs
    Little used for metagenomics...
  • 14. Why is assembly hard for metagenomes?
    Heterogeneous samples
    many different genomes
    overlap between genomes
    e.g. 16S
    Non-species-specific contigs
    http://rna.ucsc.edu/
  • 15. When could it work
    One or a few dominating species
    contigs might be species-specific
  • 16. Specialized software
    Genovo
  • 17. Specialized software
    Genovo
    Uses a 'generative probabilistic model' of read generation
    Assembler discovers 'likely sequence reconstructions under the model'
  • 18. Use your favorite assembler
    Newbler (454)
    Velvet
    Euler
    SOAPdenovo
    ...
    Tweak parameters
    e.g. higher stringency for determining overlaps
  • 19. Check contigs for
    Read depth
    GC frequency
    Tetranucleotide frequency
  • 20. Example
    Read depth
  • 21. Challenges
    Repetitive element
    DNA
    Shotgun
    reads
    Shotgun reads
    Contigs
    Collapsed contig
  • 22. Results
    Lots of pieces
    Higher read depth
    Repetitive element
    DNA
  • 23. Example
    One contig
    Log scale!
  • 24. Example
  • 25. Example
    Caulobacteraceae
    Proteobacteria
    Cyanobacteria
    Bacteroides
  • 26. Solution
    Split contigs on
    read depth
    GC%
    Use BLAST
  • 27. MetagenomicORFome Assembly
    Gene/protein-
    directed assembly
    Ye Y, Tang H. 2009. J BioinformComputBiol 7: 455-471
  • 28. Iterative read mapping and assembly
    Align reads to a single reference genome
    'Update' the reference
    based on alignment
    Align remaining reads again
    Dutilh BE, Huynen MA, Strous M. 2009. Bioinformatics 25: 2878-2881.
  • 29. Reverse metagenomics
    Leptospirillumgroup III never cultured
    shotgun metagenomics
    • nitrogen fixation gene
    • 30. GC content and read depth Leptospirillum group III
    Culturable for the first time