The benefits of environment specific curation of the public databases for taxonomic assignment
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A presentation from the Workshop: Principles, potential, and limitations of novel molecular methods in water engineering; from amplicon sequencing to omics methods. Held at the Microbial Ecology and Water Engineering 2013 (MEWE 2013) July 7 – 10, 2013.
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The benefits of environment specific curation of the public databases for taxonomic assignment
- 1. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Importance of a curated 16S database Aaron Marc Saunders MEWE 2013 CENTER FOR MICROBIAL COMMUNITIES
- 2. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY What’s in a name? Aaron Marc Saunders MEWE 2013 CENTER FOR MICROBIAL COMMUNITIES
- 3. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Agenda 1. What is taxonomic assignment? 2. Why all these names? 3. Taxonomic assignment: details 4. Ecosystem-specific curation of taxonomy
- 4. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Amplicon sequencing 1k - 1 million sequences 1000’s species sequence clustering OTU-based analysis
- 5. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Amplicon sequencing 0.1-1 million reads 1000’s OTUs sequence clustering Taxonomic assignment OTU-based analysis
- 6. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Many studies stop here… Proteobacteria Bacteroidetes Firmicutes Actinobacteria Etc… SAMPLES
- 7. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Amplicon sequencing 0.1-1 million reads 1000’s OTUs 300 genera sequence clustering Taxonomic assignment Functional data OTU-based analysis
- 8. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Functional information • Isolates • Functional 16S rRNA work – Stable isotope probing • Metagenomics • In situ studies – Microscopy – eg. inclusion bodies – Microautoradiography – Raman or NanoSIMS
- 9. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Metagenomics FA-CoA TAG α-glucan (glycogen) 2-Oxoglutarate Isocitrate Citrate Fumarate Malate Glyoxylate Pyruvate Glyceraldehyde-3P NO3 - NO3 - NO3 - NO2 - NO3 - NH4 NH4 CO2 Glutamate Glutamate NO2 - NO2 - NO2 - NO2 - Fructose-6P Trehalose Glucuronate Gluconate KDPG PH2MVPHB PHV PH2MB H+ H+ N2 N2 2-oxoglutarate Glyoxylate E.T.C. H+ H2O O2 Succinate Succinyl-CoA Oxaloacetate Propionyl-CoA Acetyl-CoA Acetyl-CoA Glycerol ? ? H+ Glucose-6P Formate Acetyl-CoA Propionyl-CoA Propionyl-CoA Acetyl-CoA b. Aerobic Simon McIllroy Competibacter metabolic model Poster Søren Karst Talk: Wed 1:15 pm Omics and Other Methods
- 10. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Amplicon sequencing 0.1-1 million reads 1000’s OTUs 300 genera sequence clustering Taxonomic assignment Functional data OTU-based analysis Species within a genera tend to be similar
- 11. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY What is Tax assignment? • Putting a name to a sequence • Phylogeny = gold-standard
- 12. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Phylogenetic analysis • Heirarchical clustering based on inferred evolutionary relationships • Requires good global alignment • Uses evolutionary model • Computationaly intensive • Requires long sequences (> 1000 nt) • Requires specific training/knowledge Source: http://www.phylogeny.fr
- 13. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY What is Tax assignment? • Putting a name to a sequence • Phylogeny = ”gold-standard” • BLAST et al. – Highest similarity match
- 14. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY NCBI BLAST as classifier ”BLAST is the wikipedia of classification” • Database is uncurated! • Often gives ambiguous result – uncultured bacterium again! • Taxonomy not based on phylogenetic analysis • BLAST uses local alignment – Good only closely-related sequences
- 15. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Taxonomic assignment Genus A Genus B Genus C Family
- 16. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Taxonomic assignment Dechloromonas Accumulibacter Zoogloea Family Genus Rhodocyclaceae
- 17. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Taxonomic assignment Dechloromonas Accumulibacter Zoogloea Class Order Family Genus Betaproteobacteria Rhodocyclales Rhodocyclaceae Accumulibacter Family Rhodocyclaceae
- 18. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Taxonomic assignment Dechloromonas Accumulibacter Zoogloea Class Order Family Genus Betaproteobacteria Rhodocyclales Rhodocyclaceae Accumulibacter Family Rhodocyclaceae
- 19. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Taxonomic assignment Dechloromonas Accumulibacter Zoogloea 93% identity 92% identity 94% identity Class Order Family Genus Betaproteobacteria Rhodocyclales Rhodocyclaceae - Family Rhodocyclaceae
- 20. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Using a ”Classifier” • Uses an existing phylogeny • lowest tax level to give significant match • On-line tools: http://www.arb-silva.dehttp://greengenes.lbl.gov http://rdp.cme.msu.edu
- 21. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY An incomplete list of methods • Word matching – RDP classifier (8-base baysien probability) • Pairwise distance to top BLAST hits – Global Alignment for Sequence Taxonomy (GAST) • BLAST with ”Lowest Common Ancestor” – PyroClust (in Pyrotagger) – MEGAN • ”Phylogenetic placement” – pplacer – RaxML – ARB parsimony insertion
- 22. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Greengenes assignment species (OTU) Class Order Family Genus OTU1 Betaproteobacteria Nitrosomonadales Nitrosomonadaceae Nitrosomonas OTU2 Nitrospira Nitrospirales Nitrospiraceae Nitrospira OTU3 Betaproteobacteria Rhodocyclales Rhodocyclaceae Propionivibrio OTU4 Betaproteobacteria Gallionellales Gallionellaceae ?? OTU5 Gammaproteobacteria ?? ?? ??
- 23. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Taxonomic assignment Dechloromonas Accumulibacter Zoogloea Class Order Family Genus Betaproteobacteria Rhodocyclales Rhodocyclaceae Accumulibacter Family Rhodocyclaceae
- 24. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Database incomplete Dechloromonas Accumulibacter Zoogloea Family Rhodocyclaceae Class Order Family Genus Betaproteobacteria Rhodocyclales Rhodocyclaceae Dechloromonas
- 25. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Taxonomy incomplete Dechloromonas Accumulibacter Zoogloea Family Rhodocyclaceae Class Order Family Genus Betaproteobacteria Rhodocyclales Rhodocyclaceae Dechloromonas
- 26. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Database incomplete Dechloromonas Accumulibacter Zoogloea Family Rhodocyclaceae Class Order Family Genus Betaproteobacteria Rhodocyclales Rhodocyclaceae ??
- 27. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Nitrotoga Class Order Family Genus Betaproteobacteria Gallionellales Gallionellaceae ?? Greengenes taxonomy:
- 28. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Curated taxonomy species (OTU) Class Order Family Genus OTU1 Betaproteobacteria Nitrosomonadales Nitrosomonadaceae Nitrosomonas OTU2 Nitrospira Nitrospirales Nitrospiraceae Nitrospira OTU3 Betaproteobacteria Rhodocyclales Rhodocyclaceae Accumulibacter OTU4 Betaproteobacteria Gallionellales Gallionellaceae Nitrotoga OTU5 Gammaproteobacteria Competibacterales Competibacteraceae Competibacter
- 29. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY genus Nitrite-oxidisers 0.3 0.2 0.1 0 Percentabundance
- 30. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY midasfieldguide.org midasfieldguide.org
- 31. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Core genera with FISH
- 32. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Activated sludge communities have a common core of abundant organisms Talk: Tuesday 2 pm ”Phosphorus removal”
- 33. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY midasfieldguide.org
- 34. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY - 39 characterised genera - 23 uncharacterised genera
- 35. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY MIDAS assignment p__Proteobacteria c__Betaproteobacteria o__Gallionellales f__Gallionellaceae g__Nitrotoga p__Proteobacteria c__Betaproteobacteria o__Nitrosomonadales f__Nitrosomonadaceae g__Nitrosomonas p__Proteobacteria c__Betaproteobacteria o__Nitrosomonadales f__Nitrosomonadaceae g__Nitrosospira p__Proteobacteria c__Betaproteobacteria o__Rhodocyclales f__Rhodocyclaceae g__Accumulibacter p__Proteobacteria c__Betaproteobacteria o__Rhodocyclales f__Rhodocyclaceae g__Dechloromonas p__Proteobacteria c__Betaproteobacteria o__Rhodocyclales f__Rhodocyclaceae g__Methyloversatilis p__Proteobacteria c__Betaproteobacteria o__Rhodocyclales f__Rhodocyclaceae g__Thauera p__Proteobacteria c__Gammaproteobacteria o__Competibacterales f__Competibacteraceae g__Competibacter Nitrifiers PAO Denitrifiers GAO
- 36. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY midasfieldguide.org midasfieldguide.org Aaron Saunders ams@bio.aau.dk mail@midasfieldguide.org
Notas del editor
- Intro
- Figure: Nitrotoga, a newly-discovered genus of nitrite-oxidisers, wasfound to becore in EPPR plants and in >1% in some. Theyarethereforelikelymaking a significantcontribution to nitriteoxidiation in theseplants.
- Figure: Nitrotoga, a newly-discovered genus of nitrite-oxidisers, wasfound to becore in EPPR plants and in >1% in some. Theyarethereforelikelymaking a significantcontribution to nitriteoxidiation in theseplants.
- Figure: Nitrotoga, a newly-discovered genus of nitrite-oxidisers, wasfound to becore in EPPR plants and in >1% in some. Theyarethereforelikelymaking a significantcontribution to nitriteoxidiation in theseplants.
- Figure: Nitrotoga, a newly-discovered genus of nitrite-oxidisers, wasfound to becore in EPPR plants and in >1% in some. Theyarethereforelikelymaking a significantcontribution to nitriteoxidiation in theseplants.