Part B: Use a Local pairwise alignment method

Now that you have finished Part A of the tutorial, this part shows how you can use a local pairwise alignment to resolve the correct mapping of the first intron in isoF and isoC.

1. Click on Pairwise view tab. Look at the left drop-down menu at the top of the view to see that the uppermost sequence (Dmel_ADH) has automatically been selected as the reference.

2. In the right drop-down menu, choose "isoF-NM_001022098.2". I The default Local: Smith Waterman method is used to create the alignment

3. To make sure all features will be displayed:
   
   
   1. Open the Tracks side-panel.
      
      
   2. Under Pairwise details, click once on the word Features.
      
      
   3. Move the Height slider to the right until you can see all the feature rows.

4. In the Pairwise view, click on the plus-sign icon next to the name Dmel_ADH to reveal the Features and Sequence Ruler tracks.

A correct alignment would show the 5’ end of the isoF sequence aligned with the two orange arrows that begin at the very start of the Dmel_ADH sequence. Instead, however, the beginnings of both the ADH reference sequence and the mRNA are shown as dimly colored context; in other words, as unaligned flanking sequence.

5. Scrolling down through the alignment observe that the 5’ end of the transcript sequence has aligned, albeit poorly, with a region beginning within the first intron. Meanwhile, a solid ungapped alignment doesn’t start until 5’ end of the second exon annotated for either isoF or isoC. (See the boxed area in the image below.)

Clearly, a local pairwise alignment was not an improvement over the first two multiple alignments tried in Part A.

Most likely, introducing a sufficient number of gaps to span the intron would reduce the score so much that the aligner can find a higher scoring segment that contains numerous short gapped regions.

Proceed to Part C: Use a Global pairwise alignment method.