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

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Importance of a curated 16S

database

Aaron Marc SaundersMEWE 2013

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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

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Amplicon sequencing

1k - 1 millionsequences 1000’s species

sequence clustering

OTU-based analysis

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Amplicon sequencing

0.1-1 million reads 1000’s OTUs

sequence clustering

Taxonomic assignment

OTU-based analysis

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Many studies stop here…

ProteobacteriaBacteroidetesFirmicutesActinobacteriaEtc…

SAMPLES

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Amplicon sequencing

0.1-1 million reads 1000’s OTUs

300 genera

sequence clustering

Taxonomic assignment

Functional data

OTU-based analysis

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Functional information

• Isolates• Functional 16S rRNA work– Stable isotope probing

• Metagenomics• In situ studies– Microscopy – eg. inclusion bodies– Microautoradiography– Raman or NanoSIMS

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Metagenomics

Simon McIllroyCompetibacter metabolic modelPoster

Søren Karst Talk: Wed 1:15 pm Omics and Other Methods

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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

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What is Tax assignment?

• Putting a name to a sequence

• Phylogeny = gold-standard

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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

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What is Tax assignment?

• Putting a name to a sequence

• Phylogeny = ”gold-standard”

• BLAST et al.– Highest similarity match

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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

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Taxonomic assignment

Genus A

Genus B

Genus C

Family

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Taxonomic assignment

Dechloromonas

Accumulibacter

Zoogloea

Family

Genus

Rhodocyclaceae

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Taxonomic assignment

Dechloromonas

Accumulibacter

Zoogloea

Class Order Family GenusBetaproteobacteria Rhodocyclales Rhodocyclaceae Accumulibacter

Family

Rhodocyclaceae

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Taxonomic assignment

Dechloromonas

Accumulibacter

Zoogloea

Class Order Family GenusBetaproteobacteria Rhodocyclales Rhodocyclaceae Accumulibacter

Family

Rhodocyclaceae

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Taxonomic assignment

Dechloromonas

Accumulibacter

Zoogloea

93% identity

92% identity

94% identity

Class Order Family GenusBetaproteobacteria Rhodocyclales Rhodocyclaceae -

Family

Rhodocyclaceae

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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

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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

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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 ?? ?? ??

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Taxonomic assignment

Dechloromonas

Accumulibacter

Zoogloea

Class Order Family GenusBetaproteobacteria Rhodocyclales Rhodocyclaceae Accumulibacter

Family

Rhodocyclaceae

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Database incomplete

Dechloromonas

Accumulibacter

Zoogloea

Family

Rhodocyclaceae

Class Order Family GenusBetaproteobacteria Rhodocyclales Rhodocyclaceae Dechloromonas

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Taxonomy incomplete

Dechloromonas

Accumulibacter

Zoogloea

Family

Rhodocyclaceae

Class Order Family GenusBetaproteobacteria Rhodocyclales Rhodocyclaceae Dechloromonas

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Database incomplete

Dechloromonas

Accumulibacter

Zoogloea

Family

Rhodocyclaceae

Class Order Family GenusBetaproteobacteria Rhodocyclales Rhodocyclaceae ??

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Nitrotoga

Class Order Family GenusBetaproteobacteria Gallionellales Gallionellaceae ??

Greengenes taxonomy:

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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

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genus

Nitrite-oxidisers

0.3

0.2

0.1

0

Perc

ent a

bund

ance

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midasfieldguide.org

midasfieldguide.org

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Core genera with FISH

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Activated sludge communities have a common core of abundant organisms

Talk: Tuesday 2 pm ”Phosphorus removal”

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midasfieldguide.org

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- 39 characterised genera- 23 uncharacterised genera

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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

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midasfieldguide.org

midasfieldguide.org

Aaron Saunders [email protected] [email protected]