Christian RinkeMicrobial GenomicsDOE, Joint Genome Institutecrinke@lbl.gov

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 Institutecrinke@lbl.gov

Introduction to ARB (From A User's Perspective)