Pairwise alignment of a short sequence to a chromosome

With the release of Lasergene 17.5 (July 2023), MegAlign Pro features a unique algorithm—adapted from one used in SeqMan NGen—that supports pairwise alignment of a short sequence (typically a cDNA) to a chromosome-length sequence. This type of alignment is impossible for other algorithms because the exons are miniscule in comparison to the large intron gaps, which range in size from 10,000 to 1 million bp. Using this new aligner, MegAlign Pro can simultaneously perform 25 pairwise alignments.

*Notes: If you need to do a more general pairwise alignment, see the topic Pairwise alignment for sequences with similar lengths. If you need to align two chromosome-length sequences together, use SeqMan NGen rather than MegAlign Pro.

To align a short sequence to a chromosome:

1. Prior to starting, make sure that your short sequences consist of extracted CDS features. You can create this type of sequence by using the Extract Features as Sequences template in SeqNinja, an application that is included with each Lasergene package.

2. Start a new project in MegAlign Pro and add one or more CDS sequences and one chromosome sequence to the project.
   
   In the Overview, the short sequence bars may barely be visible when compared to the long sequence. Upon seeing the difference in sequence lengths, MegAlign Pro automatically and correctly assumes that the long sequence is the reference and that you will be performing one or more pairwise alignments of short sequences to that reference. You do not need to manually specify the reference sequence.

3. Choose Align > Align Pairwise with Reference or click the Align button ( ). If you have followed the steps above, the Pairwise Options dialog will open and the Using menu will say Align to Chromosome: DNASTAR.
   
   
   
   In addition, you will see that the reference sequence has been marked in bold/italic blue font in the Sequences view.

*Note: If the Pairwise Options dialog does not open or if MegAlign Pro gives an error message or asks if you want to align with Mauve, this indicates that the short sequences are not extracted CDS features. Press Cancel (if that option is offered). Then start again from step 1, making sure that all short sequences consist of extracted CDS features.

4. Do not select a different option from the Using menu. However, you can adjust other parameters in this dialog if desired.
   
   
   • Mer size – The minimum length of a mer (overlapping region of a fragment read), in bases, required to be considered a match when aligning the sequences. Default is 19.
     
     
   • Minimum match – Specifies the minimum percentage of matches in an overlap required to join two sequences in the same contig. Default is 90%.
     
     
   • Temporary files – The location where temporary files will be stored. To specify or change the location, use the Browse button or open your computer’s file explorer and drag and drop the directory in the **Temporary files* text box. MegAlign Pro will remember and use the temporary file location for future alignments. Note: Choose a file location on a main drive; do not choose the desktop.
     
     
   • Estimated requirements – Displays the estimated memory and disk space required for the alignment to run without failure.
     
     
   • Available – Displays the amount of memory and free disk space on your local computer. A yellow warning triangle will appear if you do not have enough available space to meet the estimated requirements.

5. Press Align. Unless you selected specific sequences prior to step 4, a pairwise alignment will be made between each short sequence in the project and the long reference sequence. Any sequences that are in the incorrect orientation will be reverse-complemented automatically.

At the conclusion of the alignment process, MegAlign Pro creates a separate Pairwise view for each aligned pair. An empty Pairwise view is also created in case you wish to start a new comparison. To navigate between alignments, click on the Pairwise view tab and choose the desired alignment to display.

Each pairwise view shows only the part of the chromosome that the CDS sequence matched. Also, any sequence that was reverse-complemented is designated by a small red left-facing arrow next to the sequence name.

As with all pairwise views in MegAlign Pro, note that the information contained in them is transient. If you close the view, you will need to repeat the steps above in order to recalculate and view the results.