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Metagenomics and binning of viral genomes from environmental samples
- 1. Bas E. Dutilh Bacteriófagos: Aspectos básicos y moleculares. Aplicaciones Biotecnológicas Buenos Aires, June 29th 2015 Viral metagenomics
- 9. Metagenomics 1.0: database mapping • General database • Custom database – MetaHIT human gut catalogue – Omnibus of Marine Genes
- 10. Taxonomic and functional profiling Sunagawa et al. Science 2015
- 11. Metagenomics 2.0: genomes from metagenomes • Reference databases fail for most environmental metagenomes – “Dark matter”: sequences not in database • Homology searches fail for many short sequencing reads – Fast read alignment tools place upper limit on evolutionary distance • Interpretation fails for taxonomic and functional metagenomic profiles – Do functions co-occur in a genome? – To describe interactions between species you need species/genome-level resolution • Solution: assembly and binning of (draft) genomes from metagenomes
- 12. “Depending on how they are viewed, the unknowns can represent either a formidable challenge or a treasure trove for virus discovery.” Mokili, Rohwer and Dutilh Curr. Opin. Virology 2012
- 13. Unknowns in viral metagenomes Mokili, Rohwer and Dutilh Curr. Opin. Virology 2012
- 14. T1 T2 M F2 T1 T2 M F4 T1 T2 M F1 T1 T2 M F3 Reyes et al. Nature 2010
- 15. Pathway presence profiles Virome reads mapping to viral database Reyes et al. Nature 2010
- 16. 1 10 100 1000 10000 1 2 3 4 5 6 7 8 9 10 11 12 Numberofcontigs Number of samples contributing reads to contig De novo assembled contigs 6,988 de novo cross-contigs
- 17. Family 1 Family 2 Family 3 F1M F1T1 F1T2 F2M F2T1 F2T2 F3M F3T1 F3T2 F4M F4T1 F4T2 220 contigs present in ≥9 samples Family 4 Averagedepth→ Samples → 1 10 100 1000 10000 1 2 3 4 5 6 7 8 9 101112
- 18. crAssphage 97,065 bp 29.3% G+C 80 ORFs Caudovirales Dutilh et al. Nat. Comm. 2014
- 19. Dutilh et al. Nat. Comm. 2014
- 20. % Genomic position: 0 – 97,065 nt 2,906 metagenomes (940) blastn ≥95% identity ≥75 bp hit unambiguously aligned
- 21. 2,906 metagenomes 1,193 phages (861) blastn ≥95% identity ≥75 bp hit unambiguously aligned Ubiquity/abundance plot Dutilh et al. Nat. Comm. 2014
- 24. Dick et al. Genome Biol. 2009 Binning
- 25. Paired-end reads Iverson et al. Science 2012
- 26. Binning: Hi-C sequencing Beitel et al. PeerJ 2014 • Cross-link physically proximal DNA (formalin) • Restrict with enzyme • Ligate restricted sites • Sequence ligation sites
- 27. K-mer binning Ghai et al. Sci. Rep. 2011
- 28. K-mer (k=4) maps (ESOM) Abe et al. DNA Res. 2005
- 29. Depth binning Speth et al. Frontiers Microbiol 2012
- 30. Depth profile binning Wrighton et al. Science 2012 F1M F1T1 F1T2 F2M F2T1 F2T2 F3M F3T1 F3T2 F4M F4T1 F4T2
- 31. Partial nitritation/ anammox reactor (600 m3) 5.0 m 0.2 m 1.4 m 2.6 m 3.8 m untreated washed granules 1 2 3 4 5 6 7 8 washed granules DNA isolation Organic extraction Powersoil kit Organic extraction Powersoil kit Organic extraction Powersoil kit Organic extraction Powersoil kit Sample treatmentSample location DNA isolation untreated Differential sampling and DNA extraction Speth et al. submitted