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ARB basics 7.03.2007 Bánk Beszteri Stephan Frickenhaus

ARB basics 7.03.2007 Bánk Beszteri Stephan Frickenhaus. http://www.arb-home.de/. (Nucl. Acids Res. 32(4):1363-1371.). Database: aligned sequences plus sequence annotation plus trees. SSU rDNA sequence database curated by W. Ludwig the most recent: ssu_jan04_corr_opt.arb

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ARB basics 7.03.2007 Bánk Beszteri Stephan Frickenhaus

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  1. ARB basics 7.03.2007 Bánk Beszteri Stephan Frickenhaus

  2. http://www.arb-home.de/

  3. (Nucl. Acids Res. 32(4):1363-1371.)

  4. Database: aligned sequences plus sequence annotation plus trees SSU rDNA sequence database curated by W. Ludwig the most recent: ssu_jan04_corr_opt.arb But it can be anything besides SSU: aligned or unaligned homologous sequences can be imported they can be aligned trees calulated etc. Recently, also genomes

  5. Positional tree (PT) server catalogue of all oligonucleotide sequences up to 20mers in the database and the entries in which they occur (no gaps - independent of alignment) for a large DB, there are many short oligos - large storing capacity required for saving and a fast computer for establishing and updating the PT server! but it allows for fast and efficient searching for unique sequence fingerprints for that - this is why ARB is good for designing probes used for - searching for closest relatives in the automatic alignment - probe design - probe matching

  6. Name server unique identifiers for sequences are stored in the DB field name generated by ARB following internal rules particularly important when merging databases: ARB´s database comparison only compares these identifiers!

  7. Startup window

  8. menu info and some shortcuts tree mode (cursor usage) Main window

  9. Before importing sequences to be aligned: update PT server May take some time!

  10. 1 2 3 4 Sequence I/O

  11. or: Annotation

  12. given by ARB´s name server - don´t change it! Annotation

  13. Useful minutiae

  14. Important aside: saving database

  15. Aligning our sequences open the editor window

  16. search SAI: sequence associated information our sequences mode (cursor usage) The alignment editor sequences marked at the time of opening the editor are displayed

  17. Fast aligner

  18. Adding our sequences to the tree

  19. Finding them

  20. Creating alignment filters

  21. Calculating trees

  22. Viewing trees

  23. Importing / exporting / printing trees

  24. Probe design

  25. Updating PT server for probe design it might be useful to have two separate PT servers for probe design and probe matching - for design: one with only (close to) full-length sequences - for matching: one with everything available, incl. partial sequences So first, update a PT server with the complete DB (like before) Then remove partial sequences and update another PT server (next)

  26. But it is not there in our newly imported sequences: Updating the second PT server for probe design just like before, except that short sequences should be removed Species / species info:

  27. Filling in the nuc field

  28. Removing short sequences then: - update PT server (if you want to save DB at this point, don´t overwrite the complete DB!) - restart ARB

  29. choose the PT server updated with the full length sequences Probe design mark target sequences

  30. Probe design results list can be save for multiple probe design

  31. Probe matching mark a probe in the PD results window, then open PROBE MATCH choose the PT server that was updated using the complete DB (including partials)!

  32. Probe matching check taxonomy of entries with few mismatches, and the number and position of mismatches

  33. Exploring a probe when a probe is selected, it can be searched for in the alignment editor

  34. Multiprobe design if no probe can be found for a certain group, possibly a combination of multiple probes can be used to detect them specifically. For this: 1. design probes using non-stringent conditions high low

  35. Multiprobe design for the following, it is recommended to use - a DB containing only full length sequences - a PT server updated with this DB. Then:

  36. Multiprobe design

  37. Multiprobe design results

  38. Exploring probe combinations red - 1st probe green - 2nd probe mixed - both (yellow - marked!)

  39. Important things to keep in mind when working with ARB - don´t use PT servers blindly: - update PT server BEFORE importing new sequences - use separate PT servers for probe design and matching - don´t update a PT server parallel when working with ARB from several terminals - update names (esp. before merging DBs) - don´t forget to save after each succesful step :-) - read HELP a lot :-)

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