Miscellaneous MegAlign Pro questions

• Can you align thousands of amino acid (aa) polypeptide sequences with a reference and know the most common amino acid at each position?

Yes. The Sequence Logo along the bottom of the Sequence view in MegAlign Pro can be used to assess the proportions of the three most abundant amino acids at a given position. (See it in the image near the top of this post). We plan for a more quantitative solution to be added to a future release.

• How do I make a consensus sequence using MegAlign Pro?

Simply perform a multiple alignment by clicking a button. The consensus sequence appears automatically at the top of the Sequences view. It can be exported using the commands File > Export Data > Aligned Consensus or File > Export Data > Aligned Consensus without Gaps.

• Is it possible to align a subregion?

Yes. After performing an alignment, select the subrange you would like to align and choose Align > Realign Subsequences. See this topic for detailed information.

• Can MegAlign pro group and quantify exact matches in an alignment of thousands of reads, as for amplicon sequencing? (So, % of Amplicon A, % of Amplicon B, C etc.)

Lasergene does not currently have a specific workflow for this, but the following steps might provide what you need.  Perform a multiple alignment in MegAlign Pro; this will group identical sequences in the phylogenetic analysis. Then use the command Tree > Order Sequences Like Tree to help group identical and near-identical sequences in the other views.

• How can I align whole genomes for SARS-COV2 with other genomes and see the variation in just one window or figure?

We recommend performing a MAUVE alignment in MegAlign Pro for whole genome comparisons. If you are starting with sequencing data, first assemble your genome in SeqMan NGen, then export the consensus sequence for downstream analysis.

• How do I enter a large number of sequences in batches? Let’s say I am starting from a list of sequences in an Excel file.

A large list of sequences can be loaded into Lasergene software using standard methods.  The tricky part of your question is getting the sequences in Excel into a compatible format.  The data in the Excel file can look like anything so a conversion process would be different for each person or group.  I find .fasta format very simple and accepted by most molecular biology applications.  I would try to mimic .fasta, which has the following format, >Taxa Name on first line and sequence data on second line:

>Turkey

CCTTAAGG

>Fish

TCTTTAGG

>Whale

CCTGAAGC

You can modify almost any Excel sequence data file into .fasta format by using a combination of the Concatenate function in Excel to add/remove characters (like adding the “>”), and Find/Replace function in a text editor using Extended Search Mode (“\r” can be used to insert a line break between Name and Sequence).  Save as a text file and you are good to go.  Once you know how to do this the process takes a few minutes.

• What is best algorithm to use in building a tree for Avian Influenza virus Strain H9N2? How do I select sequences to develop the phylogenetic tree?

A good resource for obtaining sequence data is NCBI.  The best sequences to select would depend on your research question.  We believe you are asking what the best phylogenetic method is for generating an Avian Influenza phylogeny.  Phylogeneticists typically report more than one method.  The two most commonly used and accepted are Maximum Likelihood and Bayesian Inference.  We are currently working to include Maximum Likelihood In our MegAlign Pro application and will work on Bayesian Inference next.