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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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CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Importance of a curated 16S
database
Aaron Marc SaundersMEWE 2013
CENTER FOR MICROBIAL COMMUNITIES
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
What’s in a name?
Aaron Marc SaundersMEWE 2013
CENTER FOR MICROBIAL COMMUNITIES
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
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Amplicon sequencing
1k - 1 millionsequences 1000’s species
sequence clustering
OTU-based analysis
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Amplicon sequencing
0.1-1 million reads 1000’s OTUs
sequence clustering
Taxonomic assignment
OTU-based analysis
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Many studies stop here…
ProteobacteriaBacteroidetesFirmicutesActinobacteriaEtc…
SAMPLES
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
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
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Metagenomics
Simon McIllroyCompetibacter metabolic modelPoster
Søren Karst Talk: Wed 1:15 pm Omics and Other Methods
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
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
What is Tax assignment?
• Putting a name to a sequence
• Phylogeny = gold-standard
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
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
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
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
Genus
Rhodocyclaceae
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Taxonomic assignment
Dechloromonas
Accumulibacter
Zoogloea
Class Order Family GenusBetaproteobacteria Rhodocyclales Rhodocyclaceae Accumulibacter
Family
Rhodocyclaceae
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Taxonomic assignment
Dechloromonas
Accumulibacter
Zoogloea
Class Order Family GenusBetaproteobacteria Rhodocyclales Rhodocyclaceae Accumulibacter
Family
Rhodocyclaceae
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Taxonomic assignment
Dechloromonas
Accumulibacter
Zoogloea
93% identity
92% identity
94% identity
Class Order Family GenusBetaproteobacteria Rhodocyclales Rhodocyclaceae -
Family
Rhodocyclaceae
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
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
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 ?? ?? ??
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Taxonomic assignment
Dechloromonas
Accumulibacter
Zoogloea
Class Order Family GenusBetaproteobacteria Rhodocyclales Rhodocyclaceae Accumulibacter
Family
Rhodocyclaceae
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Database incomplete
Dechloromonas
Accumulibacter
Zoogloea
Family
Rhodocyclaceae
Class Order Family GenusBetaproteobacteria Rhodocyclales Rhodocyclaceae Dechloromonas
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Taxonomy incomplete
Dechloromonas
Accumulibacter
Zoogloea
Family
Rhodocyclaceae
Class Order Family GenusBetaproteobacteria Rhodocyclales Rhodocyclaceae Dechloromonas
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Database incomplete
Dechloromonas
Accumulibacter
Zoogloea
Family
Rhodocyclaceae
Class Order Family GenusBetaproteobacteria Rhodocyclales Rhodocyclaceae ??
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Nitrotoga
Class Order Family GenusBetaproteobacteria Gallionellales Gallionellaceae ??
Greengenes taxonomy:
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
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
genus
Nitrite-oxidisers
0.3
0.2
0.1
0
Perc
ent a
bund
ance
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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 organisms
Talk: Tuesday 2 pm ”Phosphorus removal”
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__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
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
midasfieldguide.org
midasfieldguide.org
Aaron Saunders [email protected] [email protected]