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CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Importance of a
curated 16S
database
Aaron Marc Saunders
MEWE 2013
C...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
What’s in a
name?
Aaron Marc Saunders
MEWE 2013
CENTER FOR MICROBIAL...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Agenda
1. What is taxonomic assignment?
2. Why all these names?
3. T...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Amplicon sequencing
1k - 1 million
sequences
1000’s species
sequence...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Amplicon sequencing
0.1-1 million reads 1000’s OTUs
sequence cluster...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Many studies stop here…
Proteobacteria
Bacteroidetes
Firmicutes
Acti...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Amplicon sequencing
0.1-1 million reads 1000’s OTUs
300 genera
seque...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Functional information
• Isolates
• Functional 16S rRNA work
– Stabl...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Metagenomics
FA-CoA
TAG
α-glucan
(glycogen)
2-Oxoglutarate
Isocitrat...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Amplicon sequencing
0.1-1 million reads 1000’s OTUs
300 genera
seque...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
What is Tax assignment?
• Putting a name to a sequence
• Phylogeny =...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Phylogenetic analysis
• Heirarchical clustering based on inferred
ev...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
What is Tax assignment?
• Putting a name to a sequence
• Phylogeny =...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
NCBI BLAST as classifier
”BLAST is the wikipedia of classification”
...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Taxonomic assignment
Genus A
Genus B
Genus C
Family
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Taxonomic assignment
Dechloromonas
Accumulibacter
Zoogloea
Family
Ge...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Taxonomic assignment
Dechloromonas
Accumulibacter
Zoogloea
Class Ord...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Taxonomic assignment
Dechloromonas
Accumulibacter
Zoogloea
Class Ord...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Taxonomic assignment
Dechloromonas
Accumulibacter
Zoogloea
93% ident...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Using a ”Classifier”
• Uses an existing phylogeny
• lowest tax level...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
An incomplete list of methods
• Word matching
– RDP classifier (8-ba...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Greengenes assignment
species
(OTU)
Class Order Family Genus
OTU1 Be...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Taxonomic assignment
Dechloromonas
Accumulibacter
Zoogloea
Class Ord...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Database incomplete
Dechloromonas
Accumulibacter
Zoogloea
Family
Rho...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Taxonomy incomplete
Dechloromonas
Accumulibacter
Zoogloea
Family
Rho...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Database incomplete
Dechloromonas
Accumulibacter
Zoogloea
Family
Rho...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Nitrotoga
Class Order Family Genus
Betaproteobacteria Gallionellales...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Curated taxonomy
species
(OTU)
Class Order Family Genus
OTU1 Betapro...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
genus
Nitrite-oxidisers
0.3
0.2
0.1
0
Percentabundance
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
midasfieldguide.org
midasfieldguide.org
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Core genera with FISH
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Activated sludge
communities have a
common core of
abundant organism...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
midasfieldguide.org
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
- 39 characterised genera
- 23 uncharacterised genera
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
MIDAS assignment
p__Proteobacteria c__Betaproteobacteria o__Gallione...
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
midasfieldguide.org
midasfieldguide.org
Aaron Saunders ams@bio.aau.d...
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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.

Published in: Self Improvement

The benefits of environment specific curation of the public databases for taxonomic assignment

  1. 1. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Importance of a curated 16S database Aaron Marc Saunders MEWE 2013 CENTER FOR MICROBIAL COMMUNITIES
  2. 2. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY What’s in a name? Aaron Marc Saunders MEWE 2013 CENTER FOR MICROBIAL COMMUNITIES
  3. 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. 4. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Amplicon sequencing 1k - 1 million sequences 1000’s species sequence clustering OTU-based analysis
  5. 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. 6. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Many studies stop here… Proteobacteria Bacteroidetes Firmicutes Actinobacteria Etc… SAMPLES
  7. 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. 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. 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. 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. 11. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY What is Tax assignment? • Putting a name to a sequence • Phylogeny = gold-standard
  12. 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. 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. 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. 15. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Taxonomic assignment Genus A Genus B Genus C Family
  16. 16. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Taxonomic assignment Dechloromonas Accumulibacter Zoogloea Family Genus Rhodocyclaceae
  17. 17. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Taxonomic assignment Dechloromonas Accumulibacter Zoogloea Class Order Family Genus Betaproteobacteria Rhodocyclales Rhodocyclaceae Accumulibacter Family Rhodocyclaceae
  18. 18. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Taxonomic assignment Dechloromonas Accumulibacter Zoogloea Class Order Family Genus Betaproteobacteria Rhodocyclales Rhodocyclaceae Accumulibacter Family Rhodocyclaceae
  19. 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. 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. 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. 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. 23. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Taxonomic assignment Dechloromonas Accumulibacter Zoogloea Class Order Family Genus Betaproteobacteria Rhodocyclales Rhodocyclaceae Accumulibacter Family Rhodocyclaceae
  24. 24. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Database incomplete Dechloromonas Accumulibacter Zoogloea Family Rhodocyclaceae Class Order Family Genus Betaproteobacteria Rhodocyclales Rhodocyclaceae Dechloromonas
  25. 25. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Taxonomy incomplete Dechloromonas Accumulibacter Zoogloea Family Rhodocyclaceae Class Order Family Genus Betaproteobacteria Rhodocyclales Rhodocyclaceae Dechloromonas
  26. 26. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Database incomplete Dechloromonas Accumulibacter Zoogloea Family Rhodocyclaceae Class Order Family Genus Betaproteobacteria Rhodocyclales Rhodocyclaceae ??
  27. 27. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Nitrotoga Class Order Family Genus Betaproteobacteria Gallionellales Gallionellaceae ?? Greengenes taxonomy:
  28. 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. 29. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY genus Nitrite-oxidisers 0.3 0.2 0.1 0 Percentabundance
  30. 30. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY midasfieldguide.org midasfieldguide.org
  31. 31. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Core genera with FISH
  32. 32. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY Activated sludge communities have a common core of abundant organisms Talk: Tuesday 2 pm ”Phosphorus removal”
  33. 33. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY midasfieldguide.org
  34. 34. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY - 39 characterised genera - 23 uncharacterised genera
  35. 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. 36. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY midasfieldguide.org midasfieldguide.org Aaron Saunders ams@bio.aau.dk mail@midasfieldguide.org

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