1. Vector NTI

2. Go Herd!

3. Download your sequence and open the file Click your name on my web page on the class genes page http://mupfc.marshall.edu/~murraye/good_bioinforma tics_genes.htm http://mupfc.marshall.edu/~murraye/good_bioinforma tics_genes.htm Save the file Open Windows Explore and right click the saved zip file. Select Extract Here You now should have two.abi files with a fish number. Remember your fish number, since Vector NTI will rename the files to their original abi names.

5. Open Vector NTI Contig Express

6. Import your sequences into contig express. Ignore any error messages from the program Once both of the sequence fragments have been imported into ContigExpress, select both of the fragments inside of the ContigExpress project window. Select Assemble ‡ Assemble Selected Fragments from the ContigExpress pull-down menu. The program tells you how many contigs were assembled and how long it took. Click on OK.

8. Window should look like this

9. What if it didn’t assemble? It may have too many ambiguities, usually in one of the two sequences. You will need to open them one at a time and edit them, and then try them again.

10. Editing your single sequence Add the first fragment as an abi file type. When it asks you to keep the original name, say no.

11. You will notice junk at the beginning.

13. Highlight Sequence and Cut (scissors)

14. Now you see little red arrows for cut sequences.

15. Do the same on the other side, all to the end of the sequence. Use the slide to make the peaks higher or lower.

16. Edit Individual bases Use the cursor to place it after the edited section to the left in the box with the peaks. Use the small n at the top menu and select next ambiguous base. The box must be around the base you want to change- select the highest peak. DON’T worry too much about being perfect- that is what the next step will check.

17. Continue until you have resolved all the ambiguities. Save the file with a next name.

19. Rename the Assembly by clicking on “Assembly 1” and then waiting a second and clicking again. Change the name to reflect the original fish number. Now double-click on your contig (i.e. “Contig 1”) in order to edit it. A contig editor window will open:

21. What you are seeing This window is broken up into three main panes. The upper left pane contains simple information about the contig, a textual annotated description of the contig, and a description of the fragments within the contig. The upper right pane presents a graphical representation of the contig and the way in which the fragments overlap in the contig. Also shown in the lower portion of this pane is the location of known discrepancies in the contig. These discrepancies represent nucleotides that differ between the two sequences, and will need to be resolved. The lower pane depicts the actual sequences themselves and depicts the fundamental details of the contig, showing the exact way in which the contig was constructed from the sequence fragments. Ambiguities and gaps are presented here in detail, and can be resolved manually in this lower pane.

22. The next step is to manually resolve all discrepancies in the contig. Prior to doing this, it is best to show the detailed chromatogram output from the automated sequencer. This can be done by right-clicking anywhere in the lower pane and selecting Show All chromatograms from the pop-up window.

25. Locate all ambiguity codes (depicted in red in the consensus sequence at the bottom of the lower pane, as a symbol underneath the consensus sequence and as green vertical lines in the graphics pane) and resolve the ambiguity character with the most likely alternative. In most cases, the most appropriate solution is simply to choose the unambiguous code from the other sequenced fragment in the same aligned column (where it overlaps).