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.
  1. In the right drop-down menu, choose "isoF-NM_001022098.2". I The default Local: Smith Waterman method is used to create the alignment
  1. 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. In the Options area, change the feature Rows from 3 to 5.
  1. 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.

  1. 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.

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