Transcript Introduction to ARB - Microbial Genome Program

Introduction to ARB (From A User's Perspective)
Christian Rinke
Microbial Genomics
DOE, Joint Genome Institute
[email protected]
Reasons for using molecular markers
Sort and classify organisms
Unravel evolutionary relationships
Track back the origin of organisms
Problems when using molecular markers for Phylogeny
Lateral gene transfer/ Genome plasticity
Orthologous/paralogous genes
Examples of Phylogenetic Markers
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16S rRNA
23S rRNA
Elongationfactors (EF-Tu; EF-G)
ATP-Synthase
Reg A
Hsp60
RNA-Polymerase
Gyrase
other housekeeping genes
• Presence in all organisms
• Functional constancy
• Comprehensive database
rRNA as Phylogenetic Marker
21 proteins
16S rRNA
30S
70S Ribosome
subunits
50S
34 proteins
5S rRNA
23S rRNA
Escherichia coli
16S rRNA
Primary and Secondary Structure
rRNA as Phylogenetic Marker
Advantages
• Functional constancy
• Ubiquitous distribution
• Relatively large size (information content)
• Conserved and highly variable structural elements
• Almost no lateral gene transfer
Drawbacks
• No continuous sequence change
• Multiple genes/operons
• Different species with identical 16S
rRNAs
ARB: a software environment for
sequence data
• ARB (ARBor, Latin: tree)
• UNIX based – but graphically oriented package
• Toolbox for analyzing sequence data,
emphasis on phylogeny reconstruction.
• Developed by:
Ludwig et al. (2004) ARB: a software environment for sequence data. Nucleic Acids Research. 32(4):1363-1371. doi:10.1093/nar/gkh293
Why ARB
Introduction to ARB
Why ARB
(+) Handles large databases
(+) Comprehensive package: sequence alignment, meta data,
filter calculation, phylogenetic analyses, FISH probe design
(+) available at no cost : www.arb-home.de.
(+) ready made databases available
(+/-) Unix based - SuSE Linux (Ubuntu Linux, Mac OSX)
(installation rather straight forward); ribocon
(+/-) Under development/ supporting information
Live Demo: 16S rRNA sequence of unknown organisms
Strategy:
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Retrieve
related reference sequences from
Downloadclosely
database
Genbank via BLAST > build reference data set
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Compile sequences in Fasta format in one text file
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Perform sequence alignment
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Using tree reconstruction to dentify organism
Introduction to ARB
ARB database
>>> SILVA rRNA database project:
http://www.arb-silva.de/download/arb-files/
• quality checked and regularly updated
• aligned
• small (16S/18S, SSU) and large subunit
(23S/28S, LSU) ribosomal RNA (rRNA)
sequences
• all three domains of life
Pruesse, E., C. Quast, K. Knittel, B. Fuchs, W. Ludwig, J. Peplies,
and F. O. Glöckner. SILVA: a comprehensive online resource for
quality checked and aligned ribosomal RNA sequence data
compatible with ARB. Nuc. Acids Res. 2007; Vol. 35, No. 21, p.
7188-7196
other sources:
>>> greengenes (greengenes.lbl.gov)
>>> Ribosomal Database Project (rdp.cme.msu.edu)
Live Demo
Phylogenetic
tree reconstruction
Secondary structure helix symbols
in ARB editor
Phylogenetic tree reconstruction
Phylogenetic tree reconstruction
Algorithmic (distance methods)
Neighbor Joining
Tree-searching (character based
methods)
Parsimony
Maximum Likelihood
Bayesian analysis
Phylogenetic
ARB is useful for nextgen amplicon
sequencestree reconstruction
Pre-sequencing:
• Validating primer specificity
• Primer design
Data QC:
• Viewing sequence alignments and checking taxonomic
identifications
• Resolving ‘unknowns’
• Evaluating conserved & variable gene regions
Post-processing:
• Comparative phylogenetic context
• Specific probe or primer design
Help
http://www.arb-home.de/documentation.html
http://help.arb-home.de/
http://tech.groups.yahoo.com/group/arb_users/
Ribocon:
http://www.ribocon.com/home/arb/
– ARB instillation DVD
– Workshops
Introduction to ARB
Introduction to ARB
Introduction to ARB (From A User's Perspective)
Christian Rinke
Microbial Genomics
DOE, Joint Genome Institute
[email protected]