1. Vector NTI Introduction
Flash Quiz
at the end!
Dr. Michael Rebhan 19-Jul-2007

2. What Does Vector NTI do?
Organize sequences and related information (including constructs, enzymes, oligos etc. – in a personal “database”)
Sequence annotation (what’s the function of this part? Where is domain X, and our probe?)    
Cloning: “Molecule construction” (and design)
Some basic sequence analysis (including restriction mapping, ORF finding, alignment)
Visualize ABI files (e.g. from our sequencing service) to see the electropherogram (data quality)
Many additional functions (e.g. primer design, virtual gels, 3D structure viewer, BLAST search) are available, but not as popular as the above

3. Sequence Annotation What do we know about this sequence?
what’s the function of this part? Where is domain X, our probe?

4. Cloning: circular maps, restriction analysis, planning & archiving

5. Sequence alignment: what is conserved? Where are differences?

6. The Vector NTI database
Organizing sequences and related information
(subsets, keywords, authors, …)
Personal database(s) [usually on G (backup!)]
Group-wide sharing on G: (recommended: via “archives” = one or more seq.)

7. Vector NTI Licenses Limited number of concurrent licenses
 Close it when you don’t need it !!!
Alternative: get Vector NTI 10 (static license)

8. Start Vector NTI:
>> Windows Start menu >> Informax … >> Vector NTI Explorer (usually the best starting point)

9. You should then see the Vector NTI Database Explorer (not the Windows Explorer or Internet Explorer!)
Got a license?

10. Select the DNA Part of the database
When you start with a standard Vector NTI database, it contains sequences that are needed in the official tutorials
Export a few of them as an archive. Then share it with your neighbor! in S:\vector.nti\training\sharing

11. (notice the other objects vnti manages!)
Select the DNA
Part of the
database
(notice the other objects vnti manages!)
Find Database | Backup
Tip: look at any messages carefully
Next: Sequence import from NCBI…

12. Go to NCBI (find the Pubmed link on the Intranet homepage)
Search the Nucleotide database (Genbank) for “MAPT” (microtubule-associated protein tau), limit: field “title”!
Go to NCBI (find the Pubmed link on the Intranet homepage)
training
Select all human & mouse Refseq sequences (is it the right protein??)
Then save them in a text file in Genbank format
(this can then be imported with DNA/RNA | Import | Molecule from text file …) careful: into the subset “training”!

13. Try to display the description of the sequences too!
The subset concept: one or more sequences you would like to treat as a group (not a folder!)
seq1
seq2
seq3
seq4
seq5
seq6
seq7
seq8 …
Subset 1
Subset 2
Explore the context menu
= right click! (what can I do with this sequence?)

14. Alignment
Align all!

15. Occurs in only 1 sequence? Export the alignment In MSF format …
Realign 2 sequences…
Switch on
All features
Of selected sequences
Is there a part that
Occurs in only 1 sequence?
Export the alignment
In MSF format …
Key Parameters: - gap open/extension
For difficult alignments: try T-Coffee or Muscle

16. E.g. as input into boxshade (Pasteur)  b/w publications
Close the AlignX window
Use the alignment
In MSF format …
E.g. as input into boxshade (Pasteur)  b/w publications

17. Open the mouse sequence in the “molecule viewer”
Text pane:
where is the full name of the sequence?
comments! (is the sequence reviewed?)
Feature map with links!
Note: changing toolbar (when changing panes)
Change display of feature type X …

18. Add a test feature and find it in the text/graphics/sequence panes
The importance
of the Display Setup! (e.g. restr. Enzymes)
Link Panes: use the text pane hierarchy (Feature Map) to control what is displayed!
Finding a subsequence and a feature

19. Translating selected regions
Six-frame translations

20. Reverse complement a sequence

21. Translate! New protein First make it a CDS feature
(we will analyze it…)
First make it a CDS feature
Save it, then create protein

22. EXERCISE New protein (translated from mouse MAPT mRNA)
Copy/paste
BLAST at ExPASy (just Google it): find potential homologs in human, cow (=Bos), and rat
pick SWISSPROT (curated sequences, “sp”) only!
Not MAP2 or
Other proteins!
Retrieve entries in SWISSPROT format
Align them with your new protein!
Import into Vector NTI:
Table | Import | Molecule from text file | Swissprot format
Any difference?

23. In the alignment, switch on the features of the
Mouse MAPT, and compare it to our translated protein
(use the mouse to order the sequences as shown)
Does our translated
protein contain
all the post-transl.
modification sites?

24. Examining Trace Files: Sequence Quality
S:\vector.nti\training\sequencing
Need:
example1.db.vs.sequenced.ma4 (2 sequences, 1 from db, 1 from sequencing)
example1.sequencing.ab1 (the “trace file”)
Why? N’s in our sequence, but not in the database…
Import | Molecules from archive…
Align them: where do they differ?
Assemble | ContigExpress | Open New Project
ContigExpress: Projects | Add Fragments | From ABI file (dialog: keep filename: YES!) then doubleclick to open it
Arrange both windows so you can see the alignment and the trace
Alignment: Click on the sequenced sequence
Select an area with interesting differences in the alignment (several N’s on the sequence from sequencing!), then right click and “broadcast selection”

25. Now you could edit the upper sequence in ContigExpress (using the keyboard)

26. You could now work with this edited sequence - export to FASTA
- send to vnti

27. Virtual Cloning: Construction
S:\vector.nti\training\
Need:
pUC19, pBR322 in DNA part of database
restriction enzymes: SmaI, EcoRI, AvaI
Open both molecules and then use Window | Cascade to see both at the
same time

28. Define the Fragments:
pUC19: SmaI (5’), EcoRI (3’)
Click: 5’ end
Shift+Click: 3’ end!

29. Define the Fragments:
pUC19: SmaI (5’), EcoRI (3’)
pBR322: EcoRI (5’), AvaI (3’)
Click: 5’ end
Shift+Click: 3’ end!

30. Fragments:
pUC19: SmaI (5’), EcoRI (3’)
pBR322: EcoRI (5’), AvaI (3’)
Click: 5’ end
Shift+Click: 3’ end!
Add both Fragments to the Goal list
then open the goal list, select both, and “Run” the molecule construction
The program knows they can’t fit without modification >> check the properties of AvaI and SmaI in the enzyme database (how they cut)

31. Would it work if we completely fill in the AvaI site?
Run …. That works, right?

33. Bioinformatics Support @ FMI
Help with nucleotide and protein sequence analysis (incl. personal training), function prediction, remote homologs
Help with bioinformatics tools e.g. genome browsers
Large scale data analysis & interpretation (Perl, R, …), analysis of pathways and networks
Other expertise related to bioinformatics / computational biology (basically: use of computers in biology, the scientific side of it)
Michael Rebhan, Michael Stadler

34. - Primer design flash movie (and alternatives) - Full user’s manual
- Primer design flash movie (and alternatives) - Full user’s manual! - Alternatives to vnti
- Full length? (all exons) - Tissue-specific transcripts?
- Various other tools (commonly used)

35. Genome Browser example: BLAT at UCSC
Our sequence
cDNAs
in Genbank!
Affy probesets
CpG island supports first exon
2 exons missing in our sequence (present in many transcript models, e.g. at UCSC, Refseq, and Ensembl)
The 3’UTR is longer in some sequences (but many have a much shorter one!)
A quick way to compare your sequence with other public sequences and transcript models! (click on the Genbank cDNAs to see which tissue they come from…)

36. Vector NTI links & materials
Now: the Flash Quiz!