In this section of the tutorial, you will use MegAlign Pro to align sequences and create a phylogenetic tree.

  1. Launch MegAlign Pro and choose New blank alignment project.

  1. Use File > Add Sequences and add the Exported Sequences_translated.fasta file exported from SeqNinja. When prompted to select the sequence type, click Protein.
  1. Perform the multiple alignment by choosing Align > Align Using MUSCLE. (Any method can be used in this workflow but using MUSCLE will ensure that your project will match the screenshots in this tutorial).
  1. To more easily see the locations of the three CDRs, click the Style tab on the right. If necessary, expand the Multiple Alignment section by clicking on its gray bar. Change the Comparison drop-down menu to Show only differences from reference. In this case, the “reference” is simply the consensus. The Sequences view now appears as below. The three colorful areas denote the three CDR regions.

In the next steps, you will build a phylogenetic tree to see how the sequences are related.

  1. Click on the Tree tab ().
  1. Choose Maximum Likelihood: RaxML. Keep the default settings and click OK.

MegAlign Pro displays the calculated tree.

  1. (optional) If you’d like to see the sequences in the Overview or Sequences view in the same order in which they appear in the tree, you can use Tree > Order Sequences Like Tree.
  1. (optional) To get a more accurate comparison of specific sequences, you can perform one or more pairwise alignments. Choose any two sequences from any view, then right-click and choose Align Pairwise. Keep the default BLOSUM 62 matrix (ideal for studying conservation) and press OK.

The default color scheme shows the three CDRs with colored backgrounds. Statistics appear in the header.

You will now export the first read sequence to use in the next section of this tutorial.

  1. In any view, single-click on the sequence name read1.abi-transl and choose File > Export Data > Sequences. Keep the defaults and press Save.
  1. Close MegAlign Pro. There is no need to save the project.

Proceed to Part D: Structure prediction and docking in Protean 3D.

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