Christian Rinke Microbial Genomics DOE, Joint Genome Institute [email protected] Introduction to ARB (From A User's Perspective)
Jan 31, 2016
Christian RinkeMicrobial GenomicsDOE, Joint Genome [email protected]
Introduction to ARB (From A User's Perspective)
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
• 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
Escherichia coli 16S rRNA
Primary and Secondary Structure
70S Ribosome
subunits
30S
50S
34 proteins
21 proteins
5S rRNA
23S rRNA
16S rRNA
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 (ARBor, Latin: tree)
• UNIX based – but graphically oriented package
• Toolbox for analyzing sequence data, emphasis on phylogeny reconstruction.
• Developed by:
ARB: a software environment for sequence data
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
(+) Handles large databases
(+) Comprehensive package: sequence alignment, meta data, filter calculation, phylogenetic analyses, FISH probe design
Why ARB
Introduction to ARB
(+) available at no cost : www.arb-home.de.
(+/-) Unix based - SuSE Linux (Ubuntu Linux, Mac OSX) (installation rather straight forward); ribocon
(+/-) Under development/ supporting information
(+) ready made databases available
Live Demo: 16S rRNA sequence of unknown organisms
Strategy:
-Retrieve closely related reference sequences from Genbank via BLAST > build reference data set
-Compile sequences in Fasta format in one text file
-Perform sequence alignment
-Using tree reconstruction to dentify organism
- Download database
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
Introduction to ARB
other sources: >>> greengenes (greengenes.lbl.gov)>>> Ribosomal Database Project (rdp.cme.msu.edu)
Live Demo
Phylogenetic tree reconstructionSecondary structure helix symbols in ARB editor
Phylogenetic tree reconstructionPhylogenetic tree reconstruction
Algorithmic (distance methods)
Tree-searching (character based methods)
Neighbor Joining
ParsimonyMaximum LikelihoodBayesian analysis
Phylogenetic tree reconstructionARB is useful for nextgen amplicon sequences
Pre-sequencing:• Validating primer specificity• Primer design
Data QC:• Viewing sequence alignments and checking taxonomicidentifications• Resolving ‘unknowns’• Evaluating conserved & variable gene regions
Post-processing:• Comparative phylogenetic context• Specific probe or primer design
http://help.arb-home.de/
http://tech.groups.yahoo.com/group/arb_users/
http://www.arb-home.de/documentation.html
Help
Ribocon:http://www.ribocon.com/home/arb/– ARB instillation DVD– Workshops
Introduction to ARB
Introduction to ARB
Christian RinkeMicrobial GenomicsDOE, Joint Genome [email protected]
Introduction to ARB (From A User's Perspective)