1. Christian Rinke Microbial Genomics DOE, Joint Genome Institute crinke@lbl.gov Introduction to ARB (From A User's Perspective)

2. Reasons for using molecular markers Sort and classify organisms Unravel evolutionary relationships Track back the origin of organisms

3. Problems when using molecular markers for Phylogeny Lateral gene transfer/ Genome plasticity Orthologous/paralogous genes

4. 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

5. 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

6. 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

7. 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/gkh293doi:10.1093/nar/gkh293

8. Why ARB

9. (+) 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.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

10. 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

11. 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)

12. Live Demo

13. Phylogenetic tree reconstruction Secondary structure helix symbols in ARB editor

14. Phylogenetic tree reconstruction Algorithmic (distance methods) Tree-searching (character based methods) Neighbor Joining Parsimony Maximum Likelihood Bayesian analysis

15. Phylogenetic tree reconstruction ARB is useful for nextgen amplicon sequences 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

16. 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

17. Introduction to ARB

19. Christian Rinke Microbial Genomics DOE, Joint Genome Institute crinke@lbl.gov Introduction to ARB (From A User's Perspective)